Frieda Raab, 79, a supervisor at Dr. Scholl's Co. for 20 years,died Saturday at her Northwest Side home.
Mrs. Raab emigrated from Germany in 1956 with her two youngchildren, settling in the Lincoln Square neighborhood, near Lincolnand Western.
She worked for Dr. Scholl's from 1957 to 1977. After …
HELENA, Montana (AP) — Greg Mortenson has asked a judge to throw out a lawsuit that accuses him of defrauding readers in his best-selling "Three Cups of Tea."
The Montana humanitarian's attorney says the lawsuit would subject other authors to expensive claims and stifle the free exchange of ideas.
The attorneys who filed the lawsuit argue that it should go forward because of a famous precedent. They …
Oscar nominee Viola Davis will join Denzel Washington in the revival of August Wilson's "Fences," arriving on Broadway in April.
"Fences" will open April 26 at the Cort Theatre with preview performances beginning April 14, producers Carole Shorenstein Hays and Scott Rudin announced Tuesday. The production will be directed by Kenny Leon.
Davis will play Rose, a woman, who, according to the actress, "unknowingly gives up her dreams and her power for love." Washington _ winner of Academy Awards for "Training Day" and "Glory" _ will portray her husband, the patriarchal Troy Maxson. The pivotal role of their son is …
CBS shuffled its deck of general managers Thursday - anddeliberately came up one short.
The realignment of radio station bosses in Chicago was promptedby the resignation of Harvey Pearlman after 15 years as vicepresident and general manager of WJMK-FM (104.3).
Named to succeed Pearlman at the oldies station was WeezieKramer, vice president and general manager of WMAQ-AM (670).Kramer's position at WMAQ, in turn, will be filled by RodZimmerman, who continues as vice president and general manager ofWBBM-AM (780).By putting "Newsradio 78" and "News/Sports 670" under the samecommand, CBS reduces expenses but virtually eliminates any pretenseof genuine competition …
New public health interventions are being tried all the time, yet reports of these efforts have been underrepresented in CJPH. The journal has created the "Public Health in Action" section to facilitate the description and discussion of new ways to address public health issues. It is an early notification system, a way to let readers know "what's new" in public health.
Such articles are increasingly evident in public health journals, covering everything from health service decentralization in Peru1 and primary prevention of obesity in black adolescent women2 to binational border surveillance of infectious disease3 and the development of schools of public health in Eastern …
India said Monday that Pakistani troops crossed into its part of the disputed Himalayan region of Kashmir and opened fire. Indian troops returned fire, and at least four soldiers were killed.
Indian army spokesman Brig. Gopala Krishnan Murali called the attack a "brazen violation of cease-fire."
One Indian soldier died and "three or four" Pakistani soldiers were killed in "retaliatory fire" in the Kupwara area of the region, Murali said.
Murali said that the body of one Pakistani soldier was still lying on the Indian side of the frontier and intermittent firing between troops continued.
There was no …
WEST Virginia's wisest commentators have submitted a number ofreasons for Warren McGraw's attempt to run for a full term on thestate Supreme Court while at the same time filling a partial term. Most haven't noticed that Justice McGraw finds himself in the sameposition his brother Darrell, now the attorney general, once was in.W. McGraw obviously wants to avoid the same outcome.
By winning the full term this year, W. McGraw would be free ofworry regarding eligibility - a partial term isn't enough to ensureit - for a glorious judicial pension.
Three-fourths of the prevailing salary is enough to set the mostdignified judicial nostril to quivering. If you think access to …
The Jackie Robinson West 9 and 10-year-olds will begin Section 3 Little League Baseball Tournament play Saturday when they face Lansing in a first round game at 5:30 p.m. in far south suburban Steger.
"Our kids really played well in the District 4 tournament." JRW head coach Glen Haley told the Defender. "I'm hoping they will continue to play well in the sectional tournament."
Jackie Robinson West won consecutive 9-10 year-old District 4 titles in 2002 and 2003 before getting upset by Roseland last season.
Tuesday at Jackie Robinson Stadium, they defeated Roseland, 8-7 to regain the district title.
Haley says he wasn't sure at the start of the tournament how …
President Barack Obama plans to sign an executive order Thursday to close the Guantanamo Bay detention center within a year and halt military trials of terror suspects held there, a senior administration official said.
The executive order was one of three expected imminently on how to interrogate and prosecute al-Qaida, Taliban or other foreign fighters believed to be threats to U.S. security.
The official said the president would sign the order Thursday to fulfill his campaign promise to shut down a facility that critics around the world say violates domestic and international human rights. The aide spoke on condition of anonymity because the event has not …
BEIRUT Former President Camille Chamoun, who helped leadLebanon's fight for independence and later requested the first U.S.military intervention in the Middle East, died Friday. He was 87.
A hospital statement said Mr. Chamoun, who survived fourassassination attempts, died of heart failure in Saint GeorgeHospital. He had been admitted Thursday with a heart ailment.
Camille Nimr "Tiger" Chamoun came from an ancient and prominentfamily among the Maronites, a Middle Eastern Christian community. Hewas educated at a French school in Beirut and graduated in 1923 witha law degree.
He was later elected to Lebanon's Chamber of Deputies and heldseveral …
BOSTON - It was 1 a.m. in the Red Sox clubhouse beneath the first-base seats at Fenway Park, and players were laughing, cracking jokes. Fans were giddy as they shuffled out of the old ballpark, already planning another winter of parties across New England. Those tales of long-suffering losers seem like ancient history now. Boston is streaking toward its second title in four years, beating the Rockies 2-1 Thursday night to take a 2-0 World Series lead heading to Colorado this weekend.
"I'm actually ecstatic," third baseman Mike Lowell said. "We're on the verge of winning a World Series."
Curt Schilling, still a big-game pitcher at age 40, allowed one run and four hits in 5 …
President Bush says the U.S. is developing penalties against the government of Zimbabwe. The move comes in response to what Bush says is the "blatant disregard" for democracy and human rights by Zimbabwe's longtime president.
Zimbabwe held a runoff presidential election Friday that many countries denounced. President Robert Mugabe is accused of using violence to coerce people to vote for him. …
Just because something is effortless to play live doesn't necessarily mean that it will translate to record without a fight. The simple fact is that live audiences tend to be far more forgiving and less pessimistically critical than audio tape, tired producers, and you yourself may be while recording. Besides, if you make a mess of something live it may be momentarily embarrassing, but by the time you've noticed it's far too late to do anything about it except to glare poisonously at some other musician onstage and hope the people in the front row are gullible enough to believe that it wasn't you. In the studio however, you have the luxury of screwing up as many times as you like and, if you can't seem to find something that works to your satisfaction, the dubious option of electing to preserve those screw ups for all time.
Whether it's a loss of spontaneity and objectivity in the face of sheer repetition, the permanence of your performance, or the simple fact that what you've written is not working, at some point you'll feel as if you're playing with someone else's fingers. Usually someone whose hands are encased in oversized mittens and who doesn't seem to play the piano at all -- at least not well enough to carve a place for a keyboard part in between the layers of guitar that have sprung up overnight on tracks 12 through 22.
Rather than spend a gloomy hour and a half staring stupidly at the console tearing my hair out and trying to decipher what "Alt git 3 (a) Russian pedal acoustic double" means, I try to work through songs and parts ahead of time using exercises that help to keep me relaxed, flexible in my approach, and most importantly, possessed of immediate concrete options on which to build a new part if I run into trouble. Each of these techniques stress improvisation in a framework that can be as loose or as focused as needed, and are as useful for warm ups as for composition. They also need not be confined to the exploration of musical concepts alone. I find that adding effects as well as switching between various synth options and analog keyboards in conjunction with taking new approaches to harmony, melody, rhythm, and form tends to compound their effectiveness. The more you throw into the mix, the more varied and numerous the ideas you generate will be.
As a starting point, the first of these techniques requires very little save a complete lack of intent. The idea is to sit down at your instrument of choice, place your hands on the keys randomly, and paying absolutely no attention to what you're doing, play for about ten minutes without pausing. This is not nearly as easy as it sounds. The temptation is to focus on the first reasonable idea you come up with and expand on that. Don't. The whole point of this is to slough off some of your frustration while finding a number of different ways to approach your material and not to fall in love with yourself and the first thing you play all over again, rush back into the studio and get all inflexible and pissed off when someone suggests that you may not quite have it yet. Relax. Now add some limitations to your improv. Start by playing through the basic changes and re-harmonizing them by making substitutions, adding extensions, and using alternate voicings while still maintaining as much of a lack of intent as you can muster. Don't worry too much about how dissonant some of your choices sound or the fact that you may be straying away from what the song calls for. The point of this is to break away from the established tonality, generate new ideas, and explore choices that you may have overlooked when encumbered by the existing form. Now add a simple melody over the top of your new-found changes and as you play weave your brand new melodic idea in and out of one of the previously written ones. While you work melody in one hand, continue to explore alternate harmonies and feel with the other.
This is only one of many ways to apply this exercise; try playing the melody in one hand against contrary motion improv in the other, harmonizing the existing melody with thick chord stacks in both hands, or slipping back and forth between time signatures and drastically altering the feel and tempo of the song. It really doesn't matter what the focus is as long as you're playing by instinct. Keep a small microcassette or mini disc recorder close at hand so that you can easily record any ideas that seem promising while disturbing the flow of your improv as little as possible. As you get closer to something that works for you, record enough of the idea to be able to revisit it accurately later on and then move off in another direction.
Some times a more directed approach is necessary to get deep into a song and find new options, particularly if you're dealing with extremely dense instrumentation. Strip the song down again to it's basic harmonic elements, i.e. simple triadic harmony or even intervals, and run through the form a couple of times. Now add any relevant extensions and substitutions to account for any diversions that the other musicians may be making from the basic chord structure, and run through the form again. Don't hesitate to drop notes that seem to undermine dominant melodies, or add completely new extensions or substitutions to highlight specific moments as you replay the form. It's a good idea to isolate the sections that are giving you the most grief and go after them one at a time, as well as to play along with a version of the song that's as close as possible to what you will be dealing with once you start recording in order to avoid stepping too far outside. While it's possible that you may find what you're looking for fairly swiftly by looping the entire form, sometimes you have to work through it in tiny increments to find just the right combination of voicings to build your part on.
OK, you've re-harmonized, rewritten, and come up with a brilliant part, epic in scope and perfect in subtlety. This is heart, mind, and spleen all rolled into one. Unfortunately, it still doesn't seem to work all that well, it's well past 3 a.m., the deck is playing your latest effort back endlessly like some satanic parrot with the musical equivalent of Tourette's syndrome, and you're sitting in the booth listening to your inspired, but completely inappropriate performance, and looking around desperately for something to put your fist through. At this point you have a head full of ideas, and probably have something workable; you just have to sift through it all and pull out the hooks. First, simplify your part drastically by creating as much space as possible. Start by altering the rhythmic complexity of your part and then go after the harmonic, and melodic components, decreasing their density while still maintaining the basic integrity and movement of the part. If you find that you're losing more than you can live with, take a brief trip in the other direction. Over complicate your part and move the pieces that are really important to you into new positions in the form. For instance, say there's a specific melodic passage that had previously been used as a counterpoint to the vocal line, but was just too busy to exist in the same place. Try using that same idea elsewhere as an introduction to a new section or as a transitional phrase.
This is not meant to be a gruelling process. I'm not suggesting that you over work the song until you're completely bored with it, just that you get to know it a little better and increase your repertoire of readily available ideas in general. If you have a few concrete options at your fingertips you can roll tape and take a run at all or part of the song. Take several. Chop them up and then put them back together. Overlap them. Whatever it takes. Often your first instincts are correct and all that's required to bring out the right part is to let your subconscious son it out in the pressure of the moment.
Objective: To assess whether cannabis use in adolescence and young adulthood is a contributory cause of schizophreniform psychosis in that it may precipitate psychosis in vulnerable individuals.
Method: We reviewed longitudinal studies of adolescents and young adults that examined the relations between self-reported cannabis use and the risk of diagnosis with a psychosis or of reporting psychotic symptoms. We also reviewed studies that controlled for potential confounders, such as other forms of drug use and personal characteristics that predict an increased risk of psychosis. We assessed evidence for the biological plausibility of a contributory causal relation.
Results: Evidence from 6 longitudinal studies in 5 countries shows that regular cannabis use predicts an increased risk of a schizophrenia diagnosis or of reporting symptoms of psychosis. These relations persisted after controlling for confounding variables, such as personal characteristics and other drug use. The relation did not seem to be a result of cannabis use to self-medicate symptoms of psychosis. A contributory causal relation is biologically plausible because psychotic disorders involve disturbances in the dopamine neurotransmitter systems with which the cannabinoid system interacts, as demonstrated by animal studies and one human provocation study.
Conclusion: It is most plausible that cannabis use precipitates schizophrenia in individuals who are vulnerable because of a personal or family history of schizophrenia.
(Can J Psychiatry 2006;51:556-565)
Information on funding and support and author affiliations appears at the end of the article.
Highlights
* This review summarizes recent key prospective studies that provide consistent evidence that cannabis use may be a contributory cause of psychosis.
* It also synthesizes these studies in light of other evidence on the biological plausibility of the association.
* The clinical, regulatory, and policy implications of the evidence are discussed in the companion paper to this review (Hall and Degenhardt, Can J Psychiatry 2006;51:566-74).
Key Words: cannabis, psychosis, schizophrenia, comorbidity, drug-induced psychosis, marijuana
Abbreviations used in this article
COMT catechol-O-methyltransferase
ECA Epidemiologic Catchment Area studyl
NSMHWB National Survey of Mental Health and Well-being
RR relative risk
SCL-90 Symptom Checklist, 90-item
THC delta-9-tetrahydrocannabinol
Over the past few decades, there has been growing evidence for an association between regular cannabis use and psychotic symptoms and disorders, both in the general population (1,2) and among incident cases of schizophrenia and other psychoses (3-5). This association prompts the question, Is cannabis use a contributory cause of psychosis?
It is useful to distinguish 2 ways in which cannabis use could cause psychosis (6). The strongest hypothesis is that heavy cannabis use causes a psychosis that would not have occurred in the absence of cannabis. A second, weaker hypothesis is that cannabis use is a contributory cause in the sense that it may precipitate schizophrenia in individuals vulnerable to the illness. The second hypothesis assumes that cannabis use is one factor among many others (including genetic predisposition and other unknown causes) that act together to cause schizophrenia.
These are not the only possible explanations of the association (6-15). It is possible that cannabis use and psychosis are caused by common factors that increase the risk of both, or that individuals with schizophrenia use cannabis to selfmedicate the symptoms of their disorder.
To infer that cannabis use causes psychosis in any of these ways, we need evidence of several things: an association between cannabis use and psychosis, that this association is greater than expected by chance, that cannabis use precedes psychosis, and that we can exclude plausible alternative explanations of this association (16). Evidence of the association between cannabis use and psychosis, as well as evidence that chance is an unlikely factor in this association, is readily available. Several prospective studies also show that cannabis use precedes psychosis. The difficulty lies in excluding the hypothesis that the relation between cannabis use and psychosis is due to other factors (for example, other drug use or a genetic predisposition to develop schizophrenia and subsequently use cannabis to self-medicate).
Cannabis as a Cause of Psychosis
A Specific Cannabis Psychosis
There are case reports of cannabis psychoses (17-21) describing individuals who develop psychotic disorders after using cannabis (22). These disorders have been attributed to cannabis use for combinations of the following reasons: the onset of the disorders followed the use of large quantities of cannabis; the affected individuals were confused, disorientated, and amnesic; some individuals had no personal or family history of psychosis; some individuals' symptoms remitted within days to weeks of enforced abstinence from cannabis; some individuals recovered completely and had no residual psychotic symptoms like those consistent with schizophrenia; and if the disorder recurred, it was only after the individual resumed cannabis use (6). Some commentators have criticized these case reports because they provide poor information on cannabis use and its relation to the onset of psychosis, the individual's premorbid adjustment, and the family history of psychosis (11,12). A recent retrospective study of Danish clinical registers found that most individuals with cliniciandiagnosed, cannabis-induced psychotic disorders were subsequently diagnosed with schizophrenia or another psychotic disorder (21).
Psychotic Symptoms and Cannabis Use
It is possible that cannabis use might trigger symptoms of psychosis among some users. This is distinct from a specific psychotic disorder attributable to cannabis use. Other drugs, such as amphetamines, also have the potential to trigger psychotic symptoms among some users (23). This possibility is also biologically plausible given the increasing evidence about the nature of the effects of cannabis on the brain (see discussion below). One study used an experimental design (as was used in the 1960s with amphetamines; 23) to show that intravenously administering THC to healthy volunteers without psychosis increased positive and negative psychotic symptoms in a dose-dependent way (24). It is important to note that effects on symptoms are clinically (and importantly) distinct from a psychotic disorder such as schizophrenia.
Several studies examined the relation between cannabis use and psychotic symptoms in the general population. Tien and Anthony used data from the ECA to examine correlates of reporting one or more psychotic experiences (that is, 4 types of hallucinations and 7 types of delusional beliefs), using a case-control design (2). They compared 477 individuals who reported one or more of these symptoms in a 1-year follow-up with 1818 control subjects who did not. Participants were matched for age and social and demographic characteristics. They found that daily cannabis use doubled the risk of reported psychotic symptoms (after statistical adjustment for alcohol use and psychiatric diagnoses at baseline).
Thomas reported the prevalence of psychotic symptoms among cannabis users in a random sample of individuals drawn from the electoral roll of a large city in the North Island of New Zealand (25). After using cannabis, 1 in 7 (14%) individuals reported strange, unpleasant experiences, such as hearing voices, having fears of persecution, or worrying that someone was attempting to harm them (25).
Stefanis and others reported cross-sectional relations between self-reported cannabis use and positive and negative symptoms of psychosis at age 18 years in a cohort of 3500 Greek adolescents (26). The rate of cannabis use was low, with only 6% reporting lifetime use and 0.9% reporting daily or near daily use. Nonetheless, they found positive associations between frequency of cannabis use (never, once, 2 to 4 times, 5 times or more, and daily or near daily) and 4 dimensions of psychotic experiences (paranoid, first rank, hallucinations, and grandiose experiences). These relations were not affected by controlling for other drug use or symptoms of depression. They were also stronger in individuals who reported initiation of cannabis use prior to age 15 years.
Community surveys of psychiatric disorders, such as the ECA, have reported higher rates of substance use disorders among individuals with schizophrenia (27). Nearly one-half of the patients identified in the ECA as having schizophrenia were also diagnosed with substance abuse or dependence (34% for an alcohol disorder and 28% for another drug disorder) (28). These rates were higher than those among the general population, which were 14% for alcohol disorders (29) and 6% for drug abuse (27). The most common patterns of substance use among the 231 individuals with schizophrenia in the ECA were alcohol (37%) and cannabis (23%), stimulants and hallucinogens (13%), and narcotics (10%) and sedatives (8%) (30).
The NSMHWB, conducted in Australia in 1997, included a screening questionnaire for psychotic symptoms (31). Among those under age 50 years who screened positive for a psychotic disorder, 7.8% (n = 27) met ICD-10 criteria for cannabis dependence in the past 12 months. This was 17.2% of all individuals diagnosed with cannabis dependence. A diagnosis of cannabis dependence made the chances of reporting psychotic symptoms 1.71 times more likely, after adjusting for age, affective and anxiety disorders, smoking status, and alcohol dependence (1). In the NSMHWB, 11.5% of individuals who reported being diagnosed with schizophrenia met ICD-10 criteria for a cannabis use disorder in the prior 12 months, and 21.2% met criteria for an alcohol use disorder. After adjusting for confounding variables, those who met criteria for cannabis dependence were 2.9 times more likely to report that they had been diagnosed with schizophrenia than those who did not.
Cannabis Use and Schizophrenia
Clinical Studies
In case-control studies, patients with schizophrenia are more likely to use cannabis than other psychiatric patients or control subjects without schizophrenia (32-34). The prevalence of cannabis use in patients with schizophrenia varies among studies, but it is generally higher than the rates in the general population (34,35). These variations are probably owing to differences in the sampling of patients, with younger individuals reporting higher rates of cannabis use than older individuals with chronic disorders. After alcohol and tobacco, cannabis is the most commonly used drug, and it is often used with alcohol (36,37).
Apart from finding that young men are overrepresented among cannabis users (6), as they are in the general community (38), the controlled clinical studies provide conflicting evidence on the correlates of substance abuse in schizophrenia. In some studies, cannabis users had an earlier onset of psychotic symptoms, a better premorbid adjustment, more episodes of illness, and more hallucinations (36,39-42). Other controlled studies failed to replicate some of these findings (30,43-46).
A recent clinical study adopted a novel approach to studying the relation between cannabis use and psychosis (47). In this study, 100 young individuals (49% male with an average age of 19.3 years) were identified as being at ultra high risk of psychosis on the basis of one or more criteria: schizophrenia in a first-degree relative, the presence of attenuated psychotic symptoms, or a brief, limited psychosis. Cannabis was the most commonly used drug in the 12 months preceding the assessment (35%), with 18% of participants meeting criteria for cannabis dependence in the previous year. Cannabis use, however, did not predict an increased risk of developing an acute psychosis during the follow-up period, regardless of whether cannabis use in the past year was defined as any use, frequent use, or dependent use.
Prospective Studies of Cannabis and Psychosis
The first convincing evidence that cannabis use may precipitate schizophrenia came from a 15-year prospective study of cannabis use and schizophrenia in 50 465 Swedish individuals (48). This study investigated the relation between self-reported cannabis use at age 18 years and the risk of being diagnosed with schizophrenia, as documented in the Swedish psychiatric case register, during the subsequent 15 years.
Andreasson and others found that those who tried cannabis by age 18 years were 2.4 times more likely to receive a schizophrenia diagnosis than those who had not. The risk of a schizophrenia diagnosis was related in a dose-response way to the number of times cannabis had been used by age 18 years. Compared with those who had not used cannabis, the risk of developing schizophrenia was 1.3 times higher for individuals who had used cannabis 1 to 10 times. It was 3 times higher for individuals who had used cannabis between 1 and 50 times. For individuals who had used cannabis more than 50 times, the risk of developing schizophrenia was 6 times higher, compared with those who had not used cannabis.
These risks were substantially reduced after statistical adjustment for variables related to the risk of developing schizophrenia. These included having a psychiatric diagnosis at age 18 years and having divorced parents (as an indicator of parental psychiatric disorder). Nevertheless, these relations remained statistically significant after the adjustment. Compared with individuals who never used cannabis, those who used cannabis 1 to 10 times were 1.5 times more likely to receive a schizophrenia diagnosis. Those who used cannabis 10 times or more were 2.3 times more likely to receive a schizophrenia diagnosis. Andreasson and others argued that his means that cannabis use precipitates schizophrenia in vulnerable individuals (48).
Several longitudinal studies have since supported the findings of Andreasen and others' study (Table 1). As a follow-up to the Swedish cohort study, Zammit and others reported risk over 27 years, which covers most of the risk period for the onset of psychotic disorders in a cohort that was first studied at age 18 to 20 years (49). This study improved on the earlier study in several ways: the psychiatric register provided more complete coverage of all individuals diagnosed with schizophrenia; statistical control was improved and included a larger number of potential confounding variables, such as other drug use, IQ, known risk factors for schizophrenia, and social integration; to examine the possible role of a prodrome, the study distinguished between cases that occurred in the first 5 years of the study period and those that occurred more than 5 years afterwards; and the study undertook separate analyses of individuals who only reported using cannabis at the initial assessment.
Zammit and others, as did Andreasen and others, found that cannabis use at baseline predicted an increased risk of schizophrenia during the follow-up period. They also found a dose-response relation between frequency of cannabis use at baseline and risk of schizophrenia during the follow up. They demonstrated that the relation between cannabis use and schizophrenia persisted when they statistically controlled for the effects of other drug use and other potential confounding factors, including a history of psychiatric symptoms at baseline. They estimated that 13% of schizophrenia cases could be averted if all cannabis use was prevented (that is, there was an attributable risk of 13% owing to cannabis use). The relation between cannabis use and schizophrenia was the same for the subset of the sample of individuals who only reported cannabis use at baseline, for individuals diagnosed during the first 5 years after assessment, and for individuals diagnosed during the subsequent 22 years. The relation was slightly stronger in cases observed during the first 5 years, which probably reflects the decline in cannabis use that occurs with age.
Zammit and others' findings were consistent with those of a study conducted by van Os and colleagues (50). This was a 3-year longitudinal study of the relation between self-reported cannabis use and psychosis in a community sample of 4848 individuals in The Netherlands. At baseline, subjects were assessed on cannabis and other drug use. Psychotic symptoms were assessed with a computerized diagnostic interview. Psychosis diagnoses were validated by diagnostic telephone interview with a psychiatrist or a psychologist. A consensus clinical judgment as to whether individuals had a psychotic disorder for which they needed psychiatric care was made on the basis of the interview material. van Os and others substantially replicated the findings of the Swedish cohort and extended them in several important ways: cannabis use at baseline predicted an increased risk of psychotic symptoms during the follow-up period in individuals who did not report psychiatric symptoms at baseline; there was a dose-response relation between frequency of cannabis use at baseline and risk of psychotic symptoms during the follow-up period; those who reported any psychotic symptoms at baseline were more likely to develop schizophrenia if they used cannabis than were less vulnerable individuals ; the relation between cannabis use and psychotic symptoms persisted when van Os and others statistically controlled for the effects of other drug use; and the relation between cannabis use and psychotic symptoms was stronger for individuals with more severe psychotic symptoms who were judged to need psychiatric care, van Os and others estimated that, for individuals suffering from psychosis who were judged to need psychiatric treatment, cannabis is responsible for 13% of the risk of psychotic symptoms. They also estimated that cannabis is responsible for 50% of this risk for individuals with psychotic disorders who were judged to need psychiatric treatment.
A study by Henquet and others also replicated the Swedish and Dutch studies in a 4-year follow-up of a cohort of 2437 adolescents and young adults between 1995 and 1999 in Munich (51). At baseline, subjects were assessed by a questionnaire on cannabis use and psychotic symptoms. Psychotic symptoms were assessed, in early adulthood with the Computerized Composite International Diagnostic Interview. They found a dose-response relation between self-reported cannabis use at baseline and the likelihood of reporting psychotic symptoms. As in the Dutch cohort, young individuals who reported psychotic symptoms at baseline were much more likely to experience psychotic symptoms at follow-up if they used cannabis than were their peers who did not have such a history.
Arsenault and others reported a prospective study of the relation between adolescent cannabis use and psychosis in young adults in a New Zealand birth cohort (n = 759) whose members were assessed intensively from birth on risk factors for psychotic symptoms and disorders (52). Psychotic disorders were conservatively assessed according to DSM-IV diagnostic criteria, with corroborative reports on social adjustment from family members or friends. The researchers assessed psychotic symptoms at age 11 years, before the onset of cannabis use, and distinguished between early- and late-onset cannabis use. They also examined the specificity of the association between cannabis use and psychosis by analyzing the effects of other drug use on psychotic symptoms and disorders and of cannabis use on depressive disorders.
Arsenault and others found a relation between cannabis use by age 15 years and an increased risk of psychotic symptoms by age 26 years. Controlling for other drug use did not affect the relation. After adjusting for psychotic symptoms reported at age 11 years, the relation was no longer statistically significant, which probably reflected the small number of psychotic disorders observed in the sample. The small number of participants also limited the study's ability to examine predictors of psychotic disorders at age 26 years. The measurement of cannabis and other drug use was crude (that is, none, 1 to 2 times, and 3 or more times); however, this was more likely to work against finding relations. The specificity of the effects of cannabis on psychotic symptoms was interesting: there was no relation between other drug use and psychotic disorders, and there was no relation between cannabis use and depression. There was also an interaction between psychosis risk and age of onset of cannabis use; earlier onset was more strongly related to psychosis. Arsenault and others also suggested an interaction between cannabis use and vulnerability, with a higher risk of psychosis among cannabis users who reported psychotic symptoms at age 11 years.
Caspi and colleagues subsequently analyzed data from this cohort and reported an interaction between the risk of psychosis, cannabis use, and a functional polymorphism of the COMT gene that codes for dopamine (53). They found that the individuals in the 25% of the cohort that was homozygous for a polymorphism and used cannabis were 10.9 times more likely to develop a schizophreniform disorder than their peers with the same polymorphism who did not use cannabis. In the absence of this polymorphism, young adults who used cannabis were not at any increased risk of psychosis.
Fergusson, Horwood, and Swain-Campbell conducted a longitudinal study of the relation between cannabis dependence at age 18 years and the number of psychotic symptoms reported at age 21 years in the Christchurch birth cohort in New Zealand (54). They assessed cannabis dependence according to DSM-IV criteria and psychotic symptoms according to 10 items from the SCL-90. Because this birth cohort was assessed throughout childhood and adolescence, Fergusson and colleagues were able to adjust for a large number of potential confounding variables, including self-reported psychotic symptoms at the previous assessment, other drug use, and other psychiatric disorders. They found that cannabis dependence at age 18 years predicted an increased risk of psychotic symptoms at age 21 years (RR 2.3). This association was smaller but still significant after adjustment for potential confounders (RR 1.8). More recently, Fergusson and colleagues used a more sophisticated structural equations modelling design that accounted for both observed and nonobserved confounding factors to examine the association between cannabis use and psychotic symptoms until the individuals in this cohort were aged 25 years (55). Consistent with their earlier study, they concluded that the association between cannabis and psychosis did not appear to be a result of confounding factors and that the association appeared to move from cannabis use to symptoms of psychosis, rather than vice versa.
The longitudinal studies find consistent associations between cannabis use in adolescence and the occurrence of psychotic symptoms in early adulthood, but all share a weakness: the temporal relation between cannabis use and the onset of psychotic symptoms is uncertain. Subjects in these studies are usually assessed once each year or less often and asked to report retrospectively on their cannabis use during the preceding number of years (often as crudely as the number of times cannabis was used or the number of times it was used weekly or monthly).
According to an experience sampling method, a French study by Verdoux and others provides greater detail on the temporal relation between cannabis use and psychotic symptoms (56). These investigators asked 79 college students to report their drug use and experience of psychotic symptoms at randomly selected time points, several times daily for 7 consecutive days. The students carried portable electronic devices through which ratings were prompted by randomly programed signals. The students comprised a stratified sample from a larger group; thus, high cannabis users (n = 41) and students identified as vulnerable to psychosis (n = 16) were overrepresented in the sample. Vulnerability to psychosis was determined during a personal interview and indicated by reporting one or more psychotic symptoms in the month prior to the study. Verdoux and others found a positive association between self-reported cannabis use and unusual perceptions; they found a negative association between cannabis use and hostility. That is, during periods of cannabis use, users reported more unusual perceptions and less hostility. These relations depended on vulnerability to psychosis: in vulnerable individuals cannabis use was more strongly associated with strange impressions and unusual perceptions, and its use did not decrease feelings of hostility as it did in individuals who lacked this vulnerability.
Self-Medication
The self-medication hypothesis is superficially plausible, but the evidence in its favour is not very compelling (8). The reasons that most individuals with schizophrenia use alcohol, cannabis, and other illicit drugs are similar to those of individuals who do not have schizophrenia: to relieve boredom, to provide stimulation, to feel good, and to socialize with peers (37,57,58). The drugs that are most often used by patients with schizophrenia are also those that are used by their peers: tobacco, alcohol, and cannabis.
In favour of the self-medication hypothesis is the evidence that some patients with schizophrenia report using cannabis because its euphoric effects relieve negative symptoms and depression (32,42,57,59). Dixon and others, for example, surveyed 83 patients with schizophrenia who reported that cannabis reduced anxiety and depression and that it increased a sense of calm, but at the cost of increased suspiciousness (42). Similar results were found in a recent Australian study (57).
The self-medication hypothesis has not typically been supported (51,55). Several prospective epidemiologic studies found that there was no relation between early psychotic symptoms and an increased risk of later cannabis use, which the self-medication hypothesis requires. The relation flowed from early cannabis use to psychosis rather than vice versa. Such negative results are supported by a study by Verdoux and others that used an experience sampling method to examine the temporal relation between cannabis use and psychotic symptoms (60). They found that there was no temporal relation between reporting unusual experiences and using cannabis, as would occur if self-medication were involved. One study of adolescents (younger than the above studies) was an exception. Ferdinand and others found an association (unadjusted for confounding variables) between early-onset psychotic symptoms and later cannabis use among this younger group (61). However, the authors did not discuss the possibility that there was an interaction between genetic vulnerabilities to psychosis and cannabis use as well as correlations between genetic vulnerabilities and cannabis use, which were purported by some commentators as possible factors to consider in the findings of the Ferdinand study (62). It is also possible that the unique changes of adolescence affected the nature of the relation between emerging psychotic symptoms and cannabis use (63). A later analysis of data from this cohort found more support for a causal role of cannabis use. Early cannabis use predicted psychotic symptoms after adjusting for preexisting psychopathology assessed by the Child Behavior Checklist (64).
Intervention Studies
If we could reduce cannabis use among patients with schizophrenia we could discover whether their disorders improved and whether their risk of relapse diminished. The major difficulty with this strategy is that it presupposes that we can successfully treat substance use disorders in individuals with schizophrenia. There are very few controlled outcome studies of substance abuse treatment in schizophrenia (65). Too few of these have produced large enough benefits of treatment or treated a large enough number of patients to provide an adequate chance of detecting any positive effects of abstinence on the course of disorders. Those that have been large enough have not reported results separately by diagnosis (66).
Biological Plausibility
THC, which acts on a specific cannabinoid receptor (CB^sub 1^) in the brain, is the principal psychoactive ingredient of cannabis (67). While historically the brain's dopaminergic system was thought to play an important role in psychotic disorders (68), there is increasing evidence that the cannabinoid system may be involved in schizophrenia and related psychotic disorders (69-72). CB^sub 1^ receptor knockout mice, for example, show behaviours consistent with some schizophrenia symptoms, such as reduced goal-directed activity and memory for temporal representations (70). Elevated levels of anandamide, an endogenous cannabinoid agonist, have also been found in the cerebrospinal fluid of individuals with schizophrenia (73). A recent case-control study found that individuals with schizophrenia had a greater density of CB^sub 1^ receptors in the prefrontal cortex, compared with control subjects (74).
A double-blind provocation study by D'Souza and colleagues showed that intravenous THC provokes positive and negative psychotic symptoms in a dose-dependent way in healthy volunteers (75). Caspi and others found a strong interaction between cannabis use and a common polymorphism in the COMT gene that suggests a biological basis for the relation that, if replicated, would explain why the risk of developing a psychosis after using cannabis is modest in the population as a whole (53).
The Role of Cannabis Potency
It is sometimes claimed that current cannabis is a different drug from that used in the 1970s and early 1980s (76). The United States is the only country that has analyzed the THC content of cannabis products over the past 3 decades. These data show an increase in THC content from 1.5% in the early 1970s, to 3.3% in the mid 1980s, and to 4.4% in 1998 (77). More recent European studies indicate that cultivars of cannabis with much higher THC are now being produced in The Netherlands (78,79), the use and effects of which will need to be investigated.
The increase in average THC content has overshadowed another important determinant of exposure to THC: a sharp decline in the age of initiation of cannabis use between 1970 and 2000 and a consequent increase in rates of regular cannabis use (80). These changes in patterns of use have increased both the amount of THC consumed and the duration of such consumption among adolescent cannabis users (76), thereby increasing their risk of dependence, poor educational performance, and psychotic symptoms.
Summary
There is currently good epidemiologic evidence from longitudinal studies in several different countries that regular cannabis use predicts an increased risk of schizophrenia and that this relation persists after controlling for confounding variables. There is very weak evidence that this relation is owing to self-medication. A contributory causal relation is also biologically plausible. Psychotic disorders involve disturbances in the dopamine neurotransmitter systems and cannabinoids, such as THC, increase dopamine release in the nucleus accumbens (81).
The evidence from prospective epidemiologic studies suggests that it is most likely that cannabis use precipitates schizophrenia in individuals who are vulnerable because of a personal or family history of schizophrenia. This hypothesis is consistent with the stress-diathesis model of schizophrenia (82,83) and evidence that a genetic vulnerability to psychosis increases the risk that cannabis users will develop psychosis (52,53,56,84). A vulnerability hypothesis is also consistent with the fact that the treated incidence of schizophrenia did not obviously increase during the 1970s and 1980s (85,86) when there were substantial increases in cannabis use among young adults in Australia and North America (38).
Acknowledgements
This paper is an updated version of 2 previous reviews of the evidence on cannabis and psychosis published in 2001 and 2004. Thanks to Emma Black and Amanda Roxburgh for assisting with compilation of references and proofreading the paper.
Funding and Support
An honorarium is available for each In Review series.
[Sidebar]
R�sum� : L'usage du cannabis est-il une cause concourante de la psychose?
Objectif : �valuer si l'usage du cannabis � l'adolescence et au jeune �ge adulte est une cause concourante de la psychose schizophr�niforme, en ce qu'il peut pr�cipiter la psychose chez les personnes vuln�rables.
M�thode : Nous avons examin� les �tudes longitudinales d'adolescents et de jeunes adultes qui portaient sur les relations entre l'usage autod�clar� du cannabis et le risque de recevoir un diagnostic de psychose ou de d�clarer des sympt�mes psychotiques. Nous avons aussi examin� les �tudes qui contr�laient d'�ventuelles variables confusionnelles, comme l'usage d'autres formes de drogue et des caract�ristiques personnelles qui pr�disent un risque accru de psychose. Nous avons �valu� les donn�es probantes de la plausibilit� biologique d'une relation causale concourante.
R�sultats : Les donn�es probantes de 6 �tudes longitudinales men�es dans 5 pays indiquent que l'usage r�gulier du cannabis pr�dit un risque accru d'un diagnostic de schizophr�nie ou de d�clarer des sympt�mes de psychose. Ces relations persistaient apr�s le contr�le des variables confusionnelles comme les caract�ristiques personnelles et l'usage d'autres drogues. La relation ne semblait pas r�sulter de l'usage du cannabis aux fins d'autom�dicamenter les sympt�mes de psychose. Une relation causale concourante est biologiquement plausible parce que les troubles psychotiques impliquent des perturbations des syst�mes neurotransmetteurs de la dopamine, avec lesquels le syst�me cannabino�de interagit, comme le d�montrent des �tudes animales et une �tude de provocation humaine.
Conclusion : I1 est tr�s plausible que l'usage du cannabis pr�cipite la schizophr�nie chez les personnes qui sont vuln�rables, en raison d'ant�c�dents personnels ou familiaux de schizophr�nie.
[Reference]
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[Author Affiliation]
Louisa Degenhardt, MPsych(Clinical), PhD1, Wayne Hall, PhD2
[Author Affiliation]
Manuscript received and revised March 2006, and accepted April 2006
1 Senior Lecturer, National Drug and Alcohol Research Centre, University of New South Wales, Sydney, Australia.
2 Professor, School of Population Health, University of Queensland, Queensland, Australia.
Address for correspondence: L Degenhardt, National Drug and Alcohol Research Centre, University of New South Wales, Building R3, 22-32 King Street, Sydney NSW 2052 Australia; L.Degenhardt@unsw.edu.au
ABSTRACT The finite-difference Poisson-Boltzmann methodology was applied to a series of parallel, a-helical bundle models of the designed ion channel peptide Ac-(LSSLLSL)3-CONH2. This method is able to fully describe the current-voltage curves for this channel and quantitatively explains their cation selectivity and rectification. We examined a series of energy-minimized models representing different aggregation states, side-chain rotamers, and helical rotations, as well as an ensemble of structures from a molecula dynamics trajectory. Potential energies were computed for single, permeating K+ and Cl- ions at a series of positions along a central pathway through the models. A variable-electric-field Nernst-Planck -electrodiffusion model was used, with two adjustable parameters representing the diffusion coefficients of K+ and Cl- to scale the individual ion current magnitudes. The ability of a given DelPhi potential profile to fit the experimental data depended strongly on the magnitude of the desolvation of the permeating ion. Below a pore radius of 3.8 A, the predicted profiles showed large energy barriers, and the experimental data could be fit only with unrealistically high values for the K+ and Cldiffusion coefficients. For pore radii above 3.8 A, the desolvation energies were 2kT or less. The electrostatic calculations were sensitive to positioning of the Ser side chains, with the best fits associated with maximum exposure of the Ser side-chain hydroxyls to the pore. The backbone component was shown to be the major source of asymmetry in the DelPhi potential profiles. Only two of the energy-minimizad structures were able to explain the experimental data, whereas an average of the dynamics structures gave excellent agreement with experimental results. Thus this method provides a promising approach to prediction of current-voltage curves l:rom three-dimensional structures of ion channel proteins.
INTRODUCTION
Ionic conduction through transmembrane protein pores in cell membranes is a highly regulated process in living systems (Hille, 1992). Much of this regulation is at the level of individual protein molecules, many of which show remarkable selectivity in conducting different, physiologically important metal ions. Many essential ion channel proteins have been sequenced, and their conserved sequence features have been correlated with their conduction properties (Imoto, 1993; Sather et al., 1994; Yool and Schwartz, 1991). The three-dimensional structures of several channels, including gramicidin (Roux and Karplus, 1994), porin (Weiss et al., 1991, Weiss and Schulz, 1992) and the newly solved potassium channel (Doyle et al., 1998), have been reported. However, predicting ionic currents from threedimensional protein structures is very challenging, particularly for those channels with the small pore radii (<5 A) characteristic of most ion channels associated with nerve conduction. This challenge arises because the electrostatic energies of charged species are so large, and because the electrostatic influences on permeation of charged species can become very large in confined spaces. For example, the calculated solvation energy of a K+ ion in water versus vacuum is -76.8 kcal/mol (Rashin and Honig, 1985). Even a 1% difference (-Rn in solvation energy produced by transferring the ion from bulk water to the pore would have a significant effect on permeation. In general, when an ion is transferred from an isotropic medium to one of the same dielectric constant-but which is surrounded by one of lower dielectric constant-ion transfer energies far in excess of thermal energies can result (Parsegian, 1969). The magnitude of the energies is largely dependent on the size and shape of the dielectric boundary (Parsegian, 1975).
A number of powerful computational methods are now available to calculate electrostatic potential fields from very complex, discrete distributions of charges embedded in media of discontinuous dielectric constant (Cortis and Friesner, 1997; Gilson et al., 1988; Sharp and Honig, 1990). Moreover, these methods are beginning to be applied to the calculation of ion permeation rates in static (Woolley et al., 1997) and dynamic (Hao et al., 1997; Smith and Sansom, 1997) models of natural ion channels.
Our interest has focused on understanding the ion conduction properties of LS3, a designed amphiphilic a-helical peptide with the sequence Ac-(LSSLLSL)3-CONH2. LS3 has been shown to form voltage-gated channels composed of parallel helical aggregates when incorporated into planar lipid bilayers, and single-channel conductance measurements indicate that LS3 displays both current rectification and a -10-fold selectivity for cations (kkerfeldt et al., 1995; Lear et al., 1988). The ability of LS3 to give measurable single-channel conductance for cations as large as Tris+, but not glucosammonium, suggests that LS3 has a pore size of -4 A radius, placing LS? in the group of channels with midsized pores. This group includes the cation-selective nicotinic receptor, with a 3.5-4.0-A pore radius (Cohen et al., 1992; Dwyer et al., 1980) and the anion-selective GABAA and glycine receptors, with pore radii of -3 A (Bormann et al., 1987; Fatima-Shad and Barry, 1993). The structural simplicity cf LS3 makes it a particularly attractive model for studying; the current-voltage (IV) characteristics of this class of proteins. It contains an exact heptad repeat that should induce a structurally regular multistranded coiled coil with a pore of uniform diameter. Of all elements of protein structure-in soluble as well as in membrane proteins-the coiled coil is the most accurately predicted and modeled (Crick, 1953; Dieckmann and DeGrado, 1997; Dunker and Zaleske, 1977; Kohn et al., 1997; Pauling et al., 1951; Woolfson and. Alber, 1995). In an attempt to model the cation selectivity and rectification displayed by LS3 (Kienker et al., 1994) and derivatives of this peptide (Lear et al., 1997), we previously used a continuum electrodiffusion model together with a simplified geometric model containing point charges radially arranged around a cylindrical pore. Data in symmetrical KCI solutions (indicating that the bath solutions on the two sides of the channel contain the same salt concentration) were well described by a pore with a radius of 4 A and a length of 30 A and physically reasonable adjustable parameters involving ion permeabilities, pore/wall dielectric constants, and screened end charges. However, data in asymmetric salt solutions (Kienker and Lear, 1995) were fit less satisfactorily, and the observed charge selectivity could not be explained by any of the theoretical parameters. Mathematical modeling with a kinetic model for transitions among ionoccupied and empty states allowed fitting of the data but required 13 adjustable parameters of doubtful physical significance (Kienker and Lear, 1995). Application of PoissonNernst-Planck (PNP) theory (Chen et al., 1992) fit the data reasonably well with only four parameters (Chen et al., 1997), but the best-fit value of the a-helix end charge was only 5% of its generally accepted value (Sitkoff et al., 1994). Furthermore, this theory predicted that the observed cation selectivity arose from an eightfold larger diffusion coefficient for K+ versus Cl within the channel, a result that is difficult to reconcile with molecular models of the channel. To summarize, these previous studies were able to account to some degree for the rectification and selectivity properties of LS3, but because they lacked atomic resolution information, it was not possible to analyze in any detail the structural features that give rise to channel conductance properties.
To overcome this limitation, we sought a theoretical method that would utilize the information contained in detailed molecular structures to predict channel properties. In this article we report the application ofthe finite-difference Poisson-Boltzmann (FDPB) methodology (Sharp and Honig, 1990) to compute the potential energies of K+ and Cl- ions (relative to bulk water) at discrete positions within a permeation pathway defined through the center of pores formed by a-helical bundles of LS3. Rather than focusing on a single model, we examined a series of reasonable models obtained using energy minimization or molecular dynamics calculations. Experimental results indicate that the most reasonable aggregation state for this channel is six or seven (*kerfeldt et al., 1993). Therefore, we examined models composed of five to eight helices. The potential energy profiles for each model were then calculated using Nernst-Planck (NP) electrodiffusion theory to fit experimental data with parameters to scale the magnitudes of the observed current. The structural details of how simple bundles of a-helices give rise to the specific channel properties observed for LS3 will be discussed. (We used NP instead of PNP theory because PNP does not appear to be as applicable to the single ion transit situation postulated in this work. NP and PNP are continuum electrodiffusion theories that use the same differential equation to compute current from the electrical potential. The major difference between the two theories is that in PNP the potential itself depends on the magnitude of the current; as the current increases, the density of charge inside the channel also increases, changing the potential. However, ions are discrete charges, and because we postulate single ion occupancy, it seems inconsistent to employ a theory that effectively requires the channel to retain a "memory" of the passage of previous ions.)
METHODS
Model building
The helical bundles were given a left-handed twist, using equation 1 of Dieckmann et al. (1998) with ap of either 189 A (for pentamers and hexamers) or 350 A (for heptamers and octamers; the large value of p was used to prevent excessive curvature of the helices in these bundles; Dunker and Zaleske, 1977). The parameters used in the construction of models are listed in Table 1. For each channel model, the SOAK command in Insight95 was used to place water molecules in the channel pore as well as in a 5-A-thick layer at the N- and C-termini of the channels. No water molecules were placed on the hydrophobic exteriors of the bundles. The resulting models were subjected to energy minimizations using Discover (Biosym/Molecular Simulations) and the CVFF force field (Dauber-Osguthorpe et al., 1988). The energy-minimized models displayed reasonable helix/helix packing interactions and maintained in general the helical rotations and bundle symmetries used to build the structures (Fig. 1).
The dynamic hexameric models were generated from unrestrained molecular dynamics calculations performed on a model channel of LS3 in a membrane mimetic environment (Zhong et al., 1998). Structures for DelPhi calculations were selected at 6-ps intervals after an equilibration time of -3 ns and represent the magnitude of the structural fluctuations observed for the equilibrated channel. Water and octane molecules were removed from all models before performing DelPhi calculations.
Electrostatic calculations
For each model of the LS3 ion channel, the electrostatic energy was calculated for the process of moving an ion (K+ or C1-) through the channel pore from the N-terminal side (N-side) to the C-terminal side (C-side) of the channel. The ion channel model was centered at the origin with the pore aligned along the z axis of the Cartesian coordinate system and inserted into a 34-*-thick slab of close-packed dummy atoms that simulates a membrane bilayer. Electrostatic energies were calculated using the FDPB methodology implemented in the software package DelPhi (Gilson et al., 1988; Nicholls et al., 1991; Sharp and Honig, 1990). For the calculations, the grid dimensions were 65 X 65 x 65, with a scale of - 1 grid/A. Solutions were obtained for the nonlinear Poisson-Boltzmann equation with Coulombic boundary conditions (Gilson et al., 1988`, using the multigridding method of iteration (Holst and Saied, 1993; Sharp et al., 1995). For a representative calculation, the system was mapped onto the grid in 64 different positions, and the results were averaged to obtain an estimate of the numerical precision of the calculations. The radii and charge values used are listed in Table 2; similar results were obtained from the OPLS parameter set (Jorgensen and Tirado-Rives, 1988). The solvent and protein dielectric constants were 80 and 2, respectively. The membrane slab had dimensions of 60 A X 60 A x 34 A, with a dielectric constant of 2.0. This slab separates the solvent region into two halves, with the only connection between the two being the pore through the channel.
The DelPhi program was modified to accept asymmetrical salt concentrations on the two sides of the membrane. The solvent region of the system (high dielectric) was divided into three regions by two planes, each parallel to the xy plane, that were displaced along the z axis (Fig. 2). ZN defines the z axis displacement for the plane separating the N-terminal (N-side) solvent from the pore of the channel; analogously, Zc is the z axis position for the plane between the channel pore and the C-terminal (C-side) solvent region. The values for ZN and Zc were defined by the N- and C-terminal ends, respectively, of the ion channel models. The two bulk solvent regions have the sarne high dielectric, but separate ion concentrations defined as [KCI]N and [KCl]c for the N-side and C-side regions, respectively. Within the pore, a dielectric of 80 and an ionic concentration of zero were used. (The solvent dielectric constant was 80 both inside and outside the pore. The dielectric constant of the pore water primarily affects the calculation of the ion hydration energy. Although some molecular dynamics calculations (Sansom et al., 1997b) show restricted water rotational mobility within pores such as those modeled here, such effects should not have a large influence on the solvation energy of the permeating ion. Permeant ion studies (Lear et al., 1988) indicate that the pore radius of LS3 is large enough to accommodate a K+ ion with at least one or two solvation shells of water (data not shown). It is reasonable to assume that these waters close to the K+ ion will provide as much solvation energy as they would in bulk water.) The permeating ion was treated explicitly by placing the ion at a series of positions within the pore; the ion trajectory was either assumed to be straight along the z axis or defined using the HOLE suite of programs (Smart et al., 1996). For all models examined, the two approaches gave essentially identical results.
The electrostatic energy resulting from the interaction of the permeating ion with the channel (and membrane) was computed as the sum of three components (Fig. 3). First, the ion experiences a change in solvation when moving from the bulk solvent phase to the pore of the channel. To determine the energetic consequences of this "desolvation", we computed the self-energy of the ion (Sharp and Honig, 1990) at a given position along the ion trajectory in two different dielectric environments-in bulk solvent only, as well as solvent plus the protein/membrane dielectric regions. For both self-energy calculations, only the charge associated with the permeating ion was used; partial charges of the channel atoms were set at zero. The desolvation energy is then the difference between these two self-energies (Fig. 3 A).
Contributions to the electrostatic energy also arise from the interaction of the ion with the atomic partial charges of the ion channel. These contributions wl-re broken down into two categories representing the backbone (Fig. 3 B) and side-chain atoms (Fig. 3 C). For the calculation of the backbone component, the partial charges of the backbone atoms (N, HN, CA, C and O atoms) were used, and the side-chain atom partial charges were set at zero. For the potential energy due to protein side chains interacting with the permeating ion, the Ser CB, OG, and HG atoms were assigned partial charges, with all other atoms (including all backbone and Leu side-chain atoms) set to zero.
It should be noted that the potential profiles for some of the models display an uneven, or "spiked," shape (as seen, for example, in Figs. 4, 5, and 7 A). These abrupt fluctuations in the potentials arise from very localized computed interactions. In nature, the dynamics of the system would adjust in response to these interactions and "smooth out" the potential profiles. Because we compute the integral of the potential when fitting the experimental data (Eq. 2 below), the potential profiles are smoothed by the integration process (except for the G10 hexamer, where two obviously extreme data points were removed from the calculation). For the MD hexamer, potential energy profiles were computed for each of the six structures from the dynamics trajectory and then averaged together to generate an average profile used to fit the IV data (below).
Use of DelPhi-defined potential profiles to fit current-voltage data
RESULTS
The focus of this manuscript is to evaluate a series of static and dynamic structures to determine which features give rise to efficient conduction in this class of channels. We therefore used the general approach of Dunker (Dunker and Zaleske, 1977) to mathematically generate a series of channels based on five to eight stranded idealized coiled coils. Models with systematic variations in their helix orientations and side-chain dihedrals were generated and energy minimized. The geometric features of these models and their nomenclature are presented in Table 1. A hexameric model obtained from a molecular dynamics simulation was also evaluated. The finite-difference Poisson-Boltzmann (FDPB) methodology was then used to compute the potential profiles for the channels, from which experimental IV curves could be computed by using the diffusion coefficients of K+ and CI- in the pore as the only adjustable parameters.
To determine how well the various models are able to explain the experimental data (Table 3), it is necessary to determine whether the computed adjustable parameters are reasonable. Given the dimensions of the pores, we assume that the diffusion coefficients for K+ and C1- should not be greater than those in the bulk ('K and oc, values less than or equal to 1). We therefore use three criteria to evaluate each model: 1) OK and /cl should be less than or equal to unity; 2) the r.m.s. deviation between the computed versus the experimental IV curves (up,) should approach the experimental error ( +/-0.2pA); and 3) the r.m.s. deviation between the experimental and computed reversal potentials (ero) should approach its experimental ,frror (1.7 mV). Using these criteria, the models can be grouped into four categories, as shown in Table 3. One :model from each category was selected and described in more detail below.
Group 1: models with pore radii that are too small
The first group of channels shows a very small pore radius (2.8 A average). Many models in this group provide a very reasonable fit to the experimental IV data, displaying both rectification and appropriate current magnitudes (Fig. 4 B). However, the computed values of /K and /cl are unreasonably large. The computed potential profiles display a very large barrier to ion permeability (Fig. 4 A)i; the values of PK and Pcl predict negligible partitioning of either K+ or Cl into the pore of the T10 pentamer. Given both the small pore size and the sensitivity of an ion's image charge to the pore radius (Parsegian, 1969), this is not surprising. To compensate for this unfavorable desolvation energy, unrealistically large values for the ion diffusion coefficients in the pore are computed (as indicated by 3K and /cl). All of the models in this group, which includes all of the pentaumer models, four hexamer models, and one heptamer model, generate similarly unsatisfactory values for these parameters.
Group 2: underprediction of cation selectivity
DelPhi-computed potential profil,es
One advantage of the approach used in this study is that the potential profile for each model of LS3 is computed as the sum of three separate components-the desolvation, Ser side-chain, and backbone terms-so ea,ch component can be examined separately and related to specific structural features of the models. For a given model, the desolvation term is about the same for the K+ and Cl-- ions; the Ser sidechain and backbone contributions for K+ and Cl- permeation are also similar in magnitude but opposite in sign. In this Results we will therefore only discuss the K+ profiles, although both the K+ and C1 are used in the curve-fitting procedures.
Desolvation component
The mean computed desolvation energies for K+ in the various models are shown in Fig. 9. As expected, the radius of the channel pore has a dramatic effect on the energetic penalty for moving an ion from the bulk, high-dielectric region to the anisotropic dielectric environment of the channel pore. As the pore radius becomes larger, the desolvation energy approaches zero. Models with pore radii below 3.8 A (including all pentamer models and four of the energyminimized hexamer models) have large desolvation energies, preventing effective partitioning of ions from the bath into the channel pore (Table 3).
The lack of asymmetry in the desolvation potential profiles for models with radii greater than 3.8 A (Fig. 9) reflects the uniform cylindrical shape of the pores. The three models whose computed potential profiles give reasonable fits to the experimental data (group 4, Table 3) have pore radii above 5.9 *. However, the T10 octamer model (group 2) has an average pore radius of 6.8 A but provides a poor fit, indicating that the size of the pore, although important, is not sufficient for a good fit to the experimental data.
Ser side-chain component
The mean Ser side-chain components of the potential profiles for the various models are shown in Fig. 10. In the original design of LS3, Ser was chosen as a neutral, polar residue for the lining of the pore. In this study we built different models that varied the extent to which the hydroxyl groups were exposed to solvent in the pore, as well as the specific Ser side-chain rotamer. The Ser side chains indeed appear to play a significant role in the energetics of ion permeation through the pore. Two groups of models in which the helical rotation allows maximum exposure of Ser side chains to the pore (designated GB and GM models; Table 1) show a favorable Ser side-chain component for K+ permeation. The other models have unfavorable Ser sidechain/K+ interaction energies, which actually predict a favorable partitioning of C1- rather than K+. In contrast, the most reasonable models (group 4) show favorable Ser sidechain interactions with K+, which account for much of their cation selectivity. We observe a small degree of asymmetry in the Ser component (Fig. 10), as has been discussed previously (Mitton and Sansom, 1996). However, the magnitude of this asymmetry is much smaller than the asymmetry in the backbone component and hence does not 4
Backbone component
The backbone contribution to the computed potential profiles is shown in Fig. 12 for all models. Because the peptides are all modeled as a-helices, the amide protons and carbonyl groups point toward the N-terminus and C-terminus, respectively. With the partial charges assigned to these atoms (see Table 2), a K+ ion experiences a net positive electrostatic interaction at the N-terminal half of the model and a net negative electrostatic potential on the C-terminal half (Fig. 3 C). The asymmetry in the backbone component is the only significant source of asymmetry observed in the computed potential profiles and is therefore the major source of rectification in the models. Averaging over the entire length of the channel gives a value of -0.20 + 0.05, indicating that the backbone may make a small contribution to the cation selectivity. This favorable interaction between K+ and the peptide backbone may arise from the tendency of the carbonyl groups to tilt away from the helical axis (Pauling et al., 1951), particularly when on the polar side of an amphiphilic a-helix (Barlow and Thornton, 1988; Karle and Balaram, 1990).
DISCUSSION
In this manuscript we use FDPB theory to examine a series of models of LS3 that canvas a wide range of structural space. Several facts suggest that such a continuum electrostatic approach might be appropriate for the LS3 channel: 1) we have previously demonstrated that the channel has a pore radius of at least 4 A (Lear et al., 1988). 2) Molecular dynamics calculations by Sansom (Mitton and Sansom, 1996) suggest that the relaxation times of the individual water molecules in the pore of LS3 are orders of magnitude faster than the time required for transmission of an ion through the pore. 3) Continuum electrostatic methods have been remarkably successful for the calculation of ion solvation energies (Honig et al., 1993). 4) A major energetic feature determining ion translocation rates is the dehydration energy (Parsegian, 1969); the calculation of this term is relatively insensitive to the dielectric constants of the water and protein matrix (Honig et al., 1993). Ion channels provide an important challenge for FDPB because of the extreme sensitivity of ion permeation kinetics to the structural details of the pores. In particular, we sought models that explain the permeation rates, rectification, and selectivity of LS3. Significantly, very few of the individual energy-minimized models were able to explain the experimental data, whereas the average from an ensemble of molecular dynamics structures showed excellent agreement with the data. Indeed, this ensemble was able to predict the IV curves with no adjustable parameters!
Our calculations indicate that the magnitude of the conduction through the LS3 channels is largely defined by the unfavorable dehydration energy associated with the transfer of an ion through the pore of the channel. This term reaches a maximum near the center of the bilayer, and only those models with internal radii greater than 3.8 A were able to provide reasonable fits to the data. This finding is in good agreement with the size of the channel, as experimentally estimated using variously sized, permeant cations (Lear et al., 1988). This conclusion is also consistent with the recently published structure of a bacterial potassium channel, whose pore widens to almost 10 A near the center of the channel. It is also interesting that two independently predicted models of the M2 proton channel pore region show a substantial opening of the pore near the center of the bilayer (Pinto et al., 1997; Sansom et al., 1997a). In addition, our calculations indicate that models with six to eight helices best fit the data, which is close to the number of helices inferred from the gating charge (six to seven) (Akez-feldt et al., 1993). Thus, although there was no preconceived pore radius introduced in our theory, there was good agreement between the calculations and the experimental data.
The FDPB calculations also explain the modest (-10:1) selectivity of the LS3 channels for conduction of K+. This selectivity appears to arise primarily from the interaction of the Ser side chains with the permeating ions. Interestingly, the computed energies of interaction depend on both the conformations of the Ser side chains as well as the orientation of the helices. In the models that best conform to the experimental data, we observe H-bonding between the Ser hydroxyl and the backbone carbonyl groups, as has also been described in previous MD models of LS3 (Mitton and Sansom, 1996). This arrangement positions the hydroxyl groups such that they can interact slightly better with cations than anions. However, in the absence of geometric features that stabilize a specific juxtaposition of the Ser side chains, the degree of selectivity provided by this H-bonded interaction is not great. To provide a greater degree of selectivity, it appears necessary to introduce formally charged groups (Lear et al., 1997) near the mouth of the channel, or to create a finely tuned arrangement of chelating groups within a region of smaller diameter, as in the bacterial potassium channel (Doyle et al., 1998) and gramicidin A (Roux and Karplus, 1994).
Our calculations are also able to quantitatively account for the rectification behavior of the LS3 channel, which arises from the alignment of the amide bonds in the a-helix. Sansom and co-workers have previously noted that the dipoles associated with the Ser side chains interact favorably with the amide dipoles (Mitton and Sansom, 1996). Although this effect could, in principle, modulate the magnitude of the overall helical dipole, it is not large enough to substantially attenuate the rectification associated with the a-helices.
In summary, we have developed methods that should be useful for the computation of IV curves under experimental conditions where the conductance is linear with respect to the ion concentration. This continuum theory should be applicable to single ion-occupancy channels with diameters at least as large as the LS3 pore. In addition, it will be interesting to modify this theory to evaluate channels with more restricted diameters, as the coordinates of such structures become available.
We thank Christopher Summa for computational assistance. The work was supported in part by the Materials Research Science and Engineering Center Program of the National Science Foundation (NSF) under award number DMR96-32598, NSF grant MCB95-06900, National Institutes of Health grants GM-56423 and GM-48130, and Office of Naval Research grant N00014-95-1-0220.
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[Author Affiliation]
Gregg R. Dieckmann,* James D. Lear,* Qingfeng Zhong,# Michael L. Klein,# William F. DeGrado,* and Kim A. Sharp*
[Author Affiliation]
*The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, and #Center for Molecular Modeling and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323 USA
[Author Affiliation]
Received for publication 30 June 1998 and in final form 20 October 1998. Address reprint requests to Dr. Kim A. Sharp, Department of Biochemistry and Biophysics, University of Pennsylvania, Anatomy and Chemistry Building, Room 417, 37th and Hamilton Walk, Philadelphia, PA 191046059. Tel.: 215-573-3506; Fax: 215-898-4217; E-mail: sharpk@mail.med. upenn.edu.
