Tim G. Hales

The 5-HT3B subunit is a major determinant of serotonin-receptor function

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.

The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones

1 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 μm) reversibly and dose‐dependently potentiated the amplitude of membrane currents elicited by GABA (100 μm) applied locally to bovine chromaffin cells. Intracellular application of propofol (16.8 μm) was ineffective. In rat cortical neurones and murine spinal neurones, extracellular application of 8.4 μm and 1.7–16.8 μm 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 μm) 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 μm) 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 μm) was not influenced by the benzodiazepine antagonist flumazenil (0.3 μm). A concentration of propofol (1.7 μm) 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 μm), 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 μm) was ineffective in this respect. Local application of propofol (600 μm) induced whole cell currents with a reversal potential dependent upon the Cl− gradient across the cell membrane. 6 On outside‐out membrane patches formed from bovine chromaffin cells, propofol (30 μm) induced single channels with mean chord conductances of 29 and 12 pS. The frequency of propofol channels was greatly reduced by coapplication of 1 μm bicuculline. Under identical ionic conditions, GABA (1 μm) activated single channels with mean chord conductances of 33, 16 and 10 pS. 7 Bath applied propofol (0.84–16.8 μm) dose‐dependently potentiated strychnine‐sensitive currents evoked by glycine (100 μm) in murine spinal neurones. 8 The relevance of the present results to the general anaesthetic action of propofol is discussed.

The 5-HT3 receptor--the relationship between structure and function.

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.

Voltage-Gated Na + Channel SCN5A Is a Key Regulator of a Gene Transcriptional Network That Controls Colon Cancer Invasion

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.

GABA Has Excitatory Actions on GnRH-Secreting Immortalized Hypothalamic (GT1-7) Neurons

The effects of gamma-aminobutyric acid (GABA) on clonal gonadotropin-releasing hormone (GnRH)-secreting hypothalamic (GT1-7) neurons were investigated using patch-clamp and fura-2 imaging techniques. Local application of GABA (100 microM) to GT1-7 cells voltage-clamped in the whole-cell configuration immediately increased membrane conductance and noise consistent with activation of the GABAA receptor-Cl- channel complex. Depolarization activated transient Na+ currents which were abolished by tetrodotoxin (TTX; 0.5 microM), and more sustained Ca2+ currents. Under constant current conditions, GT1-7 cells fired spontaneous action potentials, and depending on the Cl- equilibrium potential, GABA either depolarized cells, causing a rapid activation of action potentials, or hyperpolarized cells. In order to determine the effect of GABA on intact cells, the cell-attached patch configuration was used to record extracellularly. Under these conditions, application of GABA (100 microM), but not the GABAB receptor agonist baclofen (10 microM), immediately evoked multiple action potentials. Measurement of [Ca2+]i using fluorescence video microscopy and fura-2 revealed spontaneous, transient, repetitive increases in [Ca2+]i which had a periodicity ranging from 1 to 60 s. These Ca2+ oscillations were abolished by TTX (1 microM) and by the removal of extracellular Ca2+. Application of GABA (1 and 10 microM) induced an immediate increase in [Ca2+]i in all cells and increased the frequency of Ca2+ oscillations in a dose-dependent manner. The GABA-induced increase in [Ca2+]i was abolished by bicuculline and by the removal of extracellular Ca2+, and was inhibited by TTX. Baclofen (1 microM) had no effect on [Ca2+]i. These results suggest that activation of GABAA receptors has an excitatory action on GnRH-secreting immortalized hypothalamic neurons caused by a Cl(-)-dependent depolarization. GABA has been reported to increase GnRH secretion; a direct stimulatory action of the neurotransmitter on GABAA receptors of GnRH-secreting hypothalamic neurons may be responsible for this effect.

The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques

1 The characteristics of transmembrane currents evoked by 5‐hydroxytryptamine (5‐HT) in the neuroblastoma x Chinese hamster brain cell line NCB‐20 and neuroblastoma clonal cell line N1E‐115 have been studied under voltage‐clamp conditions by the whole‐cell recording and outside‐out membrane patch modes of the patch‐clamp technique. 2 In 73% of NCB‐20 cells examined (n = 221), and all N1E‐115 cells studied (n = 80), 5‐HT (10 μm) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5‐HT reversed in sign at a potential of approximately −2mV and demonstrated inward rectification. 3 The reversal potential of 5‐HT‐induced currents (E5‐HT) recorded from either NCB‐20 or N1E‐115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E‐115 cells, reducing internal K+ concentration from 140 to 20 mm produced a positive shift in E5‐HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mm was associated with a negative shift in E5‐HT of about 37 mV. A large reduction in internal Cl− concentration (from 144 to 6 mm) had little effect on E5‐HT. 4 5‐HT‐induced currents of NCB‐20 cells were unaffected by methysergide (1 μm) or ketanserin (1 μm), but were reversibly antagonized by GR38032F (0.1–1.0 nm) with an IC50 of 0.25 nm. GR 38032F (0.3 nm) reduced 5‐HT‐induced currents in N1E‐115 cells to approximately 26% of their control value. 5 On outside‐out membrane patches excised from both NCB‐20 and N1E‐115 cells, 5‐HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nm) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5‐HT (1 μm) in N1E‐115 cells suggests that 5‐HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside‐out membrane patches to 5‐HT cannot at present be resolved into clear single channel events.

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

Radioligand binding and patch‐clamp techniques were used to study the actions of γ‐aminobutyric acid (GABA) and the general anaesthetics propofol (2,6‐diisopropylphenol), pentobarbitone and 5α‐pregnan‐3α‐ol‐20‐one on rat α1 and β3 GABAA receptor subunits, expressed either alone or in combination. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [35S]‐tert‐butyl bicyclophosphorothionate ([35S]‐TBPS) (<0.03 pmol mg−1 protein). GABA (100 μm) applied to whole‐cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents. The membranes of cells expressing β3 cDNAs bound [35S]‐TBPS (∼1 pmol mg−1 protein). However, the binding was not influenced by GABA (10 nm–100 μm). Neither GABA (100 μm) nor picrotoxin (10 μm) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABAA receptors or spontaneously active ion channels. GABA (10 nm–100 μm) modulated [35S]‐TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μm–1 mm) also dose‐dependently activated inward currents with an EC50 of 9 μm recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV. Propofol (10 nm–100 μm), pentobarbitone (10 nm–100 μm) and 5α‐pregnan‐3α‐ol‐20‐one (1 nm–30 μm) modulated [35S]‐TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μm), pentobarbitone (1 mm) and 5α‐pregnan‐3α‐ol‐20‐one (10 μm) also activated currents recorded from cells expressing α1β3 receptors. Propofol (1 μm–1 mm) and pentobarbitone (1 mm) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α‐pregnan‐3α‐ol‐20‐one (10 μm) failed to activate detectable currents. Propofol (100 μm)‐activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1− equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μm). 5α‐Pregnan‐3α‐ol‐20‐one (100 nm) enhanced propofol (100 μm)‐evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α‐pregnan‐3α‐ol‐20‐one (10 μm) caused only a modest facilitation (to 128±12% of control) of propofol (100 μm)‐evoked currents mediated by β3 homomers. Propofol (3–100 μm) activated α1β3 and β3 receptors in a concentration‐dependent manner. For both receptor combinations, higher concentrations of propofol (300 μm and 1 mm) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge’ current on cessation of propofol (300 μm and 1 mm) application. Surge currents were also evident following pentobarbitone (1 mm) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α‐pregnan‐3α‐ol‐20‐one (10 μm) to cells expressing α1β3 receptors. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

Evidence for Expression of Heteromeric Serotonin 5-HT3 Receptors in Rodents

Abstract : The gene and cDNAs that encode a novel subunit of rodent serotonin 5‐HT3 receptors were isolated from mouse and rat tissues. Each of the new rodent subunits shares 40% amino acid identity with the rat 5‐HT3A subunit and 73% identity with the human 5‐HT3B subunit. Despite a relatively low level of structural conservation, sequence analysis and functional studies suggest that the new rodent subunits are orthologues of the human 5‐HT3B subunit. In common with homologous human receptors, rat heteromeric 5‐HT3 receptors displayed a substantially larger single‐channel conductance than homomeric 5‐HT3A receptors. In addition, the rat heteromeric receptors were less sensitive to antagonism by tubocurarine. However, in contrast to human heteromeric receptors, those of the rat displayed pronounced inward rectification of both the whole‐cell and single‐channel current amplitudes. Transcripts of the mouse 5‐HT3A and 5‐HT3B subunits are coexpressed in several cell lines that possess endogenous 5‐HT3 receptors. In addition, treatment of rat PC12 cells with nerve growth factor induced expression of both subunit mRNAs, with a similar time course for accumulation of each transcript. The combination of functional data and expression patterns is consistent with the existence of heteromeric 5‐HT3 receptors in rodent neurons.

Functional Properties of CaV1.3 (α1D) L-type Ca2+ Channel Splice Variants Expressed by Rat Brain and Neuroendocrine GH3 Cells

Ca2+ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca2+ entry through activation of pertussis toxin-sensitive G proteins leading to activation of K+channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca2+ channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three α1(α1A, α1C, and α1D), four β, and one α2-δ subunit in rat pituitary GH3 cells; α1B and α1Stranscripts were absent. GH3 cells express multiple alternatively spliced α1D mRNAs. Many of the α1D transcript variants encode “short” α1D (α1D-S) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other α1 subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (α1D-L). We cloned and expressed the predominant α1D-S transcript variants in rat brain and GH3 cells and their αlD-L counterpart in GH3 cells. Unlike α1A channels, α1D channels exhibited current-voltage relationships similar to those of native GH3 cell Ca2+channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced α1Dtranscripts form functional Ca2+ channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of α1D-S subunit, is not sufficient to confer sensitivity to inhibitory G proteins.

Potentiation, activation and blockade of GABAA receptors of clonal murine hypothalamic GT1‐7 neurones by propofol

1 The actions of GABA and the intravenous general anaesthetic propofol (2,6‐düsopropylphenol) on GABAA receptors of self‐replicating GT1‐7 hypothalamic neurones were investigated by the patch clamp technique. 2 GABA (1 μm‐1 mM) dose‐dependently activated inward currents with an EC50 of 27 μm, recorded from whole cells voltage‐clamped at —60 mV. GABA (100 μm)‐activated currents reversed at the Cl− equilibrium potential. 3 Propofol (0.1 −100 μm) dose‐dependently potentiated GABA (100 μm)‐evoked currents with an EC50 of 5 μm. 4 In the absence of GABA, propofol (10 μm‐1 mM) activated small inward currents with a reversal potential similar to the CI− equilibrium potential. The peak current amplitudes activated by propofol were only 31% of those activated by GABA in the same cells. 5 Like GABA (100 μm)‐activated currents, propofol (100 μm)‐activated currents were inhibited by the GABAA receptor antagonist, bicuculline (10 μm) and were abolished by Zn2+ (100 μm). 6 Propofol (10, 30 and 100 μm) dose‐dependently activated currents in the absence of GABA. However, the peak amplitude of currents activated by propofol declined with concentrations > 100 μm. The cessation of application of a high dose of propofol (1 mM) was associated with a current ‘surge’. 7 The surge current, seen after application of propofol (1 mM), had a reversal potential similar to the CI− equilibrium potential. The ratio between peak current amplitude in the presence of propofol (1 mM) and surge current amplitude after propofol application, were not dependent on holding potential. Thus, it is unlikely that the surge current represents reversal of a voltage‐dependent block of GABAA receptors by propofol. 8 The amplitude of the surge current exceeded the amplitude of the initial propofol (1 mM)‐evoked current following brief applications, but declined after prolonged applications of the drug. 9 The observed modulatory actions of propofol may be due to separate potentiation, activation and inhibitory sites for this anaesthetic agent on GT1‐7 cell GABAA receptors.