The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9-17 pS). Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current-voltage relationship that differ from those of homomeric receptors. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current-voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

No abstract

A common feature of general anaesthetic agents is their ability to potentiate neuronal inhibition through GABA(A) (gamma-aminobutyric acid) receptors. At concentrations relevant to clinical anaesthesia, these agents cause a dramatic stimulation of the chloride currents that are evoked by the binding of the natural ligand, GABA. Although there is widespread evidence that the sensitivity of GABA(A) receptors to anaesthetic agents is heterogeneous, the structural basis of these differences is largely unknown. Variations in subunit composition can have profound effects on the sensitivity of GABA(A) receptors to modulatory agents such as benzodiazepines. However, strict subunit specificity has not been demonstrated for the potentiating effects of anaesthetic agents. Here we describe a new class of human GABA(A) receptor subunit (epsilon) that can assemble with alpha- and beta-subunits and confer an insensitivity to the potentiating effects of intravenous anaesthetic agents. The epsilon-subunit also abolishes the normal outward rectification of recombinant receptors in which it assembles. The expression pattern of this subunit in the brain suggests a new target for manipulation of neuronal pathways within the basal ganglia.

Voltage-gated Na + channels (VGSC) have been implicated in the metastatic potential of human breast, prostate, and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Na v 1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiologic recordings. Na + channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by small interfering RNAs (siRNA) specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, with strong Na v 1.5 protein staining found in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process, and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion. Cancer Res; 70(17); 6957-67. ©2010 AACR.

University of Dundee, Dundee DD1 9SY1 The interaction of the intravenous general anaesthetic propofol (2,6-diisopropylphenol) with the GABAA receptor has been investigated in voltage-clamped bovine chromaffin cells and rat cortical neurones in cell culture. Additionally, the effects of propofol on the glycine and GABAA receptors of murine spinal neurones were determined. 2 Propofol (1.7-16.8 uM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by GABA (100puM) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 pM) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4pM and 1.7-16.8/pM propofol respectively produced a potentiation of GABA-evoked currents qualitatively similar to that seen in the bovine chromaffin cell. 3 The potentiation by propofol (1.7,UM) was not associated with a change in the reversal potential of the GABA-evoked whole cell current. On outside-out membrane patches isolated from bovine chromaffin cells, propofol (1.7,UM) had little or no effect on the GABA single channel conductances, but greatly increased the probability of the GABA-gated channel being in the conducting state. 4 The potentiation of GABA-evoked whole cell currents by propofol (1.7 pM) was not influenced by the benzodiazepine antagonist flumazenil (0.3 pM). A concentration of propofol (1.7/pM) that substantially potentiated GABA currents had little effect on currents induced by the activation of the GABAA receptor by pentobarbitone (1 mM). 5 Bath application of propofol (8.4-252 pM), to bovine chromaffin cells voltage clamped at -60 mV, induced an inward current associated with an increase in membrane current noise on all cells sensitive to GABA. Intracellular application of propofol (16.8,UM) was ineffective in this respect. Local application of propofol (600pM) induced whole cell currents with a reversal potential dependent upon the CF-gradient across the cell membrane.6 On outside-out membrane patches formed from bovine chromaffin cells, propofol (30M) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 CM bicuculline. Under identical ionic conditions, GABA (1 pM) activated single channels with mean chord conductances of 33, 16 and 10pS.7 Bath applied propofol (0.84-16.8 juM) dose-dependently potentiated strychnine-sensitive currents evoked by glycine (100,Mm) in murine spinal neurones.8 The relevance of the present results to the general anaesthetic action of propofol is discussed.

The 5-hydroxytryptamine type-3 (5-HT3) receptor is a cation-selective ion channel of the Cys-loop superfamily. 5-HT3 receptor activation in the central and peripheral nervous systems evokes neuronal excitation and neurotransmitter release. Here, we review the relationship between the structure and the function of the 5-HT3 receptor. 5-HT3A and 5-HT3B subunits are well established components of 5-HT3 receptors but additional HTR3C, HTR3D and HTR3E genes expand the potential for molecular diversity within the family. Studies upon the relationship between subunit structure and the ionic selectivity and single channel conductances of 5-HT3 receptors have identified a novel domain (the intracellular MA-stretch) that contributes to ion permeation and selectivity. Conventional and unnatural amino acid mutagenesis of the extracellular domain of the receptor has revealed residues, within the principle (A-C) and complementary (D-F) loops, which are crucial to ligand binding. An area requiring much further investigation is the subunit composition of 5-HT3 receptors that are endogenous to neurones, and their regional expression within the central nervous system. We conclude by describing recent studies that have identified numerous HTR3A and HTR3B gene polymorphisms that impact upon 5-HT3 receptor function, or expression, and consider their relevance to (patho)physiology.

We describe the presence of functional GABA(A) receptors on T cells. GABA inhibited anti-CD3 and antigen-specific T cell proliferation in vitro in a dose-dependent manner that was 1) mimicked by the GABA(A) receptor agonist muscimol (but not the GABA(B) receptor agonist baclofen), 2) blocked by GABA(A) receptor antagonists and a GABA(A) receptor Cl- channel blocker (picrotoxin) and 3) enhanced by pentobarbital. These data suggest that GABA(A) receptors mediate this immune inhibition and that these receptors can be modulated in a similar fashion to their neuronal counterparts. Finally, GABA inhibited DTH responses in vivo. Thus, pharmacological modulation of GABA(A) receptors may provide new approaches to modulate T cell responses in inflammation and autoimmune disease.

There are five families of vertebrate Cys loop receptors as follows: the nicotinic acetylcholine receptor (nAChR), 3 the 5-hydroxytryptamine type 3 receptor (5-HT 3 R), the zinc-activated ion channel, the ␥-aminobutyric acid type A receptor, and the strychnine-sensitive glycine receptor (1, 2). Structural analysis by cryo-EM of tubular crystals prepared from the Torpedo marmorata electric organ revealed that five subunits combine in nAChRs, forming a rosette around the central ion channel (3). The second transmembrane (M2) domain of each subunit participates in lining the channel pore, and collectively they present a hydrophobic constriction adjacent to what is traditionally believed to be the rate-limiting portion of the ion conduction pathway that controls single channel conductance (␥) and ionic selectivity (2, 4).The homomeric 5-HT 3A receptor is unique among Cys loop receptors, having a ␥ below the resolution of single channel recording, estimated by variance analysis to be in the femtosiemen range. The incorporation of the 5-HT 3B subunit into human heteromeric 5-HT 3A/B receptors increases ␥ to 16 pS, enabling direct observation of events by single channel recording from outside-out patches (5). The use of chimeric 5-HT 3A -5-HT 3B constructs and site-directed mutagenesis revealed a critical role of three arginine residues within the MA helix of the M3-M4 cytoplasmic loop in determining ␥ (6, 7). This has prompted speculation that the MA helix may participate in the control of ␥ in other Cys loop receptors (4,7,8). In support of this interpretation, cryo-EM analysis revealed portals within the Torpedo nAChR formed, in part, by the MA helices of adjacent subunits that may participate in the ion conduction pathway (9).In this study we investigated the influence of the 5-HT 3A subunit's MA helix Arg-432 (Ϫ4Ј), Arg-436 (0Ј), and Arg-440 (4Ј) residues in the control of ␥. We investigated the effect of introducing arginine into the equivalent locations within the nAChR ␣ 4 and ␤ 2 subunits. Our data confirm the critical role of MA Ϫ4Ј, 0Ј, and 4Ј residues in controlling ␥ of 5-HT 3A receptors and support the hypothesis that the MA helix also forms part of the ion conduction pathway of nAChRs. Our functional data provide support for the existence of cytoplasmic portals depicted in the 4 Å structural model of the nAChR (10). EXPERIMENTAL PROCEDURES DNA Constructs and Transient Transfection of Subunit cDNAs-cDNAs encoding rat wild-type (WT) nAChR ␣ 4 and ␤ 2 subunits (Dr. J. M. Boulter, Department of Psychiatry and Biobehavioral Sciences, UCLA), human WT 5-HT 3A subunits, and mutant nAChR and 5-HT 3A subunits were cloned into pGW1 (11). Point mutations were introduced using standard molecular biological techniques (7). All cDNAs were sequenced to confirm fidelity. Transfection of tsA-201, or HEK-293 cells, with subunit cDNAs, at equimolar ratios when appropriate, was performed by either the calcium phosphate precipitation method or electroporation (400 V, infinite resistance, 125 microfarads) using a Bio-Rad gene ...