Complex psychiatric disorders are resistant to whole-genome analysis due to genetic and etiological heterogeneity. Variation in resting electroencephalogram (EEG) is associated with common, complex psychiatric diseases including alcoholism, schizophrenia, and anxiety disorders, although not diagnostic for any of them. EEG traits for an individual are stable, variable between individuals, and moderately to highly heritable. Such intermediate phenotypes appear to be closer to underlying molecular processes than are clinical symptoms, and represent an alternative approach for the identification of genetic variation that underlies complex psychiatric disorders. We performed a whole-genome association study on alpha (α), beta (β), and theta (θ) EEG power in a Native American cohort of 322 individuals to take advantage of the genetic and environmental homogeneity of this population isolate. We identified three genes (SGIP1, ST6GALNAC3, and UGDH) with nominal association to variability of θ or α power. SGIP1 was estimated to account for 8.8% of variance in θ power, and this association was replicated in US Caucasians, where it accounted for 3.5% of the variance. Bayesian analysis of prior probability of association based upon earlier linkage to chromosome 1 and enrichment for vesicle-related transport proteins indicates that the association of SGIP1 with θ power is genuine. We also found association of SGIP1 with alcoholism, an effect that may be mediated via the same brain mechanisms accessed by θ EEG, and which also provides validation of the use of EEG as an endophenotype for alcoholism.alcoholism | electroencephalogram | endophenotype | genetics | whole-genome association G enetic studies of behavior and psychiatric disorders are hampered by etiologic heterogeneity of these complex phenotypes. Addiction vulnerability arises from both internalizing (emotional) and externalizing (dyscontrol) behavioral dimensions (1), and both of these broad aspects of behavior are strongly influenced by early life trauma and other gene/environment interactions (2). Etiologic heterogeneity dilutes power to detect genetic effects, and is a reason for failures to detect and replicate genome-wide associations (GWAS) in complex disorders. Increasing sample size does not remove underlying heterogeneity and can introduce additional confounds. In neuropsychiatry, these considerations have led to the use of intermediate phenotypes (or endophenotypes) that are heritable, relevant to disease, and have good measurement properties and assay variation more closely related to gene function (3) as surrogates to probe the underlying biology of complex disorders.

All experiments were performed on rat isolated desheathed superior cervical ganglia maintained in Krebs solution containing amino‐oxyacetic acid (10 μm) at 25°C. Influx rates of γ‐amino‐n‐butyric acid (GABA) were measured by incubating ganglia in 0.5 μm [3H]‐GABA for 30 minutes. Influx was inhibited by 50% on adding 14.3 μm unlabelled GABA, 59.2 μm β‐alanine (BALA) or 424 μm β‐amino‐n‐butyric acid (BABA). Efflux of [3H]‐GABA into non‐radioactive solution superfused over ganglia previously incubated for 60 min in 1 μm [3H]‐GABA was measured. The mean resting efflux rate coefficient (k) was 0.64 ±0.05 × 10−3 min−1. Addition of high concentrations of unlabelled GABA, BABA or BALA to the superfusing solution increased k by (maximally) 3.6–4.3 times; half‐maximal increases occurred at the following concentrations: GABA, 16 μm; BALA, 85 μm; BABA, 606 μm. Replacement of external Na+ with Li+ or TRIS increased the resting value of k and inhibited acceleration by external amino acids. Prior incubation in 1 μm [3H]‐GABA with 1 mm unlabelled GABA increased resting k 1.5 times;, but did not alter the peak rate coefficient produced by external amino acids. Neuronal depolarization produced by the amino acids was measured with surface electrodes. Preincubation in 1 mm GABA for 60 min potentiated low‐amplitude responses to BALA or BABA but not those to GABA or 3‐aminopropanesulphonic acid (a potent agonist with low affinity for the GABA carrier). Omission of external Na+ reduced responses to BABA but increased those to GABA. Incubation in 1 mm GABA for 60 min (as required to potentiate BABA or BALA actions) increased the amount of GABA in the tissue from 0.21 to 0.73 mmol/kg wet weight. Autoradiographs in which labelled GABA was used indicated that uptake into neuroglial cells was responsible for this accumulation. It is suggested that: (i) BALA and BABA are substrates for the inward GABA carrier responsible for GABA entry into ganglionic glial cells; (ii) they accelerate efflux by inhibiting carrier‐mediated re‐accumulation of effluent GABA by the glial cells; (iii) interstitial GABA concentrations are thereby increased to a level capable of depolarizing adjacent neurones; and (iv) this, rather than direct GABA‐receptor activation, accounts for the depolarization produced by low concentrations of BALA and BABA. Potentiation of their depolarizing action after pre‐incubation in 1 mm GABA is suggested to result from the increased amount of intracellular GABA available for release, and is quantitatively compatible with this increase; inhibition in Na+‐free solution is due to their inability to inhibit re‐accumulation of GABA under these conditions. A model for the action of carrier substrates is described in an Appendix. Calculations based thereon yield increments in interstitial GABA concentration in the presence of carrier substrates compatible with those determined experimentally (up to 1 μm at rest or 3.4 μm after pre‐incubation in GABA).

KI, 28 /UM), fi-alanine (KI, 55 /LM), y-amino-fl-hydroxybutyric acid (KI, 220 /LM), fl-amino-n-butyric acid (KI, 708 jzm), 3-aminopropanesulphonic acid (KI, 832 /IM) and taurine (KI > 1 mM). Uptake was not depressed by 1 mM-glycine, a-alanine, leucine, serine, methionine or ac-amino-iso-butyric acid.8. Radioactively labelled methionine, leucine, glycine, serine, fi-alanine and taurine (concentrations < 5SUM) were also taken up by ganglia. Of these, only uptake of fl-alanine and taurine were significantly depressed by 1 mm-GABA.9. Autoradiographs confirmed that [3H]GABA and [3H]fl-alanine were taken up predominantly into extraneuronal sites (presumed to be neuroglial cells). Methio-

In their recent article in PNAS, Das and Krantz (1) attempt to relate the previously described (2, 3) alternate conductance substate of the anthrax toxin protective antigen (PA 63 ) channel to the allosteric helix compression model (4). Although we do not intend to discuss the differences between this model and the extended-chain Brownian ratchet model (5), we believe that there are two experimental observations that should be specifically addressed.First, the authors hypothesize that the observed small-conductance substate (2, 3) is related to the two states of the PA 63 channel, one clamped empty state and one unclamped empty dilated state, with the dilation occurring at the ϕ-clamp (1). Although this interpretation could be relevant, we believe the arguments provided to support it are indirect. In addition to analyzing the so-called long-lived and short-lived closures induced by the lethal factor (LF) peptides, the authors could have also performed a direct statistical comparison of these closures when the channel is in one of the two conductance states.Second, the authors attribute the fast-flickering closures of PA 63 , that were previously characterized as a voltage-independent (2) 1/f-noise process (3), to the interaction of "a contaminant small molecule, buffer ion, or peptide" that "momentarily binds and occupies the ϕ-clamp" (1). To support this statement, the authors cite two articles (2, 6) where cationic compounds were shown to reversibly block the channel. We tested the "contaminant" hypothesis performing single PA 63 recordings in two different solutions: 1 M 99.0% pure KCl prepared using Milli-Q water and 1 M 99.99995% pure KCl prepared using deionized HPLCgrade water. The power spectral density analysis of PA 63 current fluctuations showed no statistically significant difference between these solutions (Fig. 1A). To rule out the possibility of contaminants resulting from protein or lipid addition, we made the measurements while gradually replacing the bathing KCl solutions with fresh KCl; however, no decrease in the density of the current power spectra was detected (Fig. 1B).The idea that the anthrax toxin channel structural dynamics can be directly studied on a single-channel level is intriguing. However, consistency in the interpretation of the reported data (1-3) is essential. Unfortunately, the authors (1) do not mention the fact that the heptameric PA 63 was also reported to insert directly in the higher conductance substate, switching to the lower conductance state only on occasion (3). Moreover, 7+β-cyclodextrins were shown to reversibly block both types of PA 63 insertions with statistically indistinguishable rate constants (3). The authors report that the difference between the conductance states was threefold greater for the F427A mutant (1), but do not mention that the probability of finding PA 63 in the "clamped empty state" was higher in phosphatidylserine compared with phosphatidylcholine membranes, with the substate amplitude being close to 30% of PA 63 conductance (3).In addition...