Mutational analysis of molecular requirements for the actions of general anaesthetics at the γ-aminobutyric acid(A )receptor subtype, α1β2γ2

BACKGROUND: Amino acids in the β subunit contribute to the action of general anaesthetics on GABA(A )receptors. We have now characterized the phenotypic effect of two β subunit mutations in the most abundant GABA(A )receptor subtype, α1β2γ2. RESULTS: The β2(N265M) mutation in M2 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, while the direct actions of propofol, etomidate and alphaxalone were impaired. The β2(M286W) mutation in M3 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, whereas the direct action of propofol and etomidate, but not of alphaxalone, was impaired. CONCLUSIONS: We found that the actions of general anaesthetics at α1β2(N265M)γ2 and α1β2(M286W)γ2 GABA(A )receptors are similar to those previously observed at α2β3(N265M)γ2 and α2β3(M286W)γ2 GABA(A )recpetors, respectively, with the notable exceptions that the direct action of propofol was decreased in α1β2(M286W)γ2 receptors but indistinguishable form wild type in α2β3(M286W)γ2 receptors and that the direct action of alphaxalone was decreased in α1β2(N265M)γ2 but not α2β3(N265M)γ2 receptors and indistinguishable form wild type in α1β2(M286W)γ2 receptors but increased in α2β3(M286W)γ2 receptors. Thus, selected phenotypic consequences of these two mutations are GABA(A )receptor subtype-specific

TRPM Channels Mediate Zinc Homeostasis and Cellular Growth during Drosophila Larval Development

SummaryTRPM channels have emerged as key mediators of diverse physiological functions. However, the ionic permeability relevant to physiological function in vivo remains unclear for most members. We report that the single Drosophila TRPM gene (dTRPM) generates a conductance permeable to divalent cations, especially Zn2+ and in vivo a loss-of-function mutation in dTRPM disrupts intracellular Zn2+ homeostasis. TRPM deficiency leads to profound reduction in larval growth resulting from a decrease in cell size and associated defects in mitochondrial structure and function. These phenotypes are cell-autonomous and can be recapitulated in wild-type animals by Zn2+ depletion. Both the cell size and mitochondrial defect can be rescued by extracellular Zn2+ supplementation. Thus our results implicate TRPM channels in the regulation of cellular Zn2+ in vivo. We propose that regulation of Zn2+ homeostasis through dTRPM channels is required to support molecular processes that mediate class I PI3K-regulated cell growth

Characterization of a proton-activated, outwardly rectifying anion channel

Anion channels are present in every mammalian cell and serve many different functions, including cell volume regulation, ion transport across epithelia, regulation of membrane potential and vesicular acidification. Here we characterize a proton-activated, outwardly rectifying current endogenously expressed in HEK293 cells. Binding of three to four protons activated the anion permeable channels at external pH below 5.5 (50% activation at pH 5.1). The proton-activated current is strongly outwardly rectifying, due to an outwardly rectifying single channel conductance and an additional voltage dependent facilitation at depolarized membrane potentials. The anion channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) rapidly and potently inhibited the channel (IC(50): 2.9 μm). Flufenamic acid blocked this channel only slowly, while mibefradil and amiloride at high concentrations had no effect. As determined from reversal potential measurements under bi-ionic conditions, the relative permeability sequence of this channel was SCN(−)> I(−)> NO(3)(−)> Br(−)> Cl(−). None of the previously characterized anion channel matches the properties of the proton-activated, outwardly rectifying channel. Specifically, the proton-activated and the volume-regulated anion channels are two distinct and separable populations of ion channels, each having its own set of biophysical and pharmacological properties. We also demonstrate endogenous proton-activated currents in primary cultured hippocampal astrocytes. The proton-activated current in astrocytes is also carried by anions, strongly outwardly rectifying, voltage dependent and inhibited by DIDS. Proton-activated, outwardly rectifying anion channels therefore may be a broadly expressed part of the anionic channel repertoire of mammalian cells

Transient Receptor Potential Melastatin 1 (TRPM1) Is an Ion-conducting Plasma Membrane Channel Inhibited by Zinc Ions*

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents

Biomarkers for the effects of antipsychotic drugs in healthy volunteers

Studies of novel antipsychotics in healthy volunteers are traditionally concerned with kinetics and tolerability, but useful information may also be obtained from biomarkers of clinical endpoints. A useful biomarker should meet the following requirements: a consistent response across studies and antipsychotics; a clear response of the biomarker to a therapeutic dose; a dose–response relationship; a plausible relationship between biomarker, pharmacology and pathogenesis. In the current review, all individual tests found in studies of neuroleptics in healthy volunteers since 1966 were progressively evaluated for compliance with these requirements. A MedLine search yielded 65 different studies, investigating the effects of 23 different neuroleptics on 101 different (variants of) neuropsychological tests, which could be clustered into seven neuropsychological domains. Subjective and objective measures of alertness, and of visual-visuomotor-auditory and motor skills were most sensitive to antipsychotics, although over half of all the studies failed to show statistically significant differences from placebo. The most consistent effects were observed using prolactin response and saccadic eye movements, where 96% and 83% of all studies resp. showed statistically significant effects. The prolactin inducing dose equivalencies relative to haloperidol of 19 different antipsychotic agents correlated with the lowest recommended daily maintenance dose (r2 = 0.52). This relationship could reflect the clinical practice of aiming for maximum tolerated levels, or it could represent a common basis behind prolactin release and antipsychotic activity (probably D2-receptor antagonism). The number of tests used in human psychopharmacology appears to be excessive. Future studies should look for the most specific and sensitive test within each of the domains that are most susceptible to neuroleptics