High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor.

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.

Ethanol stabilizes the open state of single 5-hydroxytryptamine(3A)(QDA) receptors.

Ethanol enhancement of 5-hydroxytryptamine (5-HT)(3A) receptor-mediated responses may have important consequences in the intoxicating and addictive properties of ethanol. Although the exact mechanism is unknown, ethanol-mediated enhancement of 5-HT(3) receptor current has been proposed to occur due to stabilization of the open-channel state. It has not been possible to directly measure the open state of the channel due to the extremely low single-channel conductance of 5-HT(3A) channels. Recently, three arginine residues within the large intracellular loop of the 5-HT(3A) subunit were substituted by their equivalent residues (glutamine, aspartate, and alanine) of the 5-HT(3B) subunit to produce a 5-HT(3A)(QDA) subunit that forms functional homomeric channels exhibiting a measurable single-channel conductance. Using whole-cell rapid-agonist application techniques and the cell-attached single-channel recording configuration, we examined human 5-HT(3A)(QDA) receptors expressed in human embryonic kidney 293 cells. The agonist sensitivity, macroscopic kinetics, and modulation by ethanol were similar between mutant and wild-type channels, suggesting the substitutions had not altered these channel structure-function properties. The open time histogram for single-channel events mediated by 5-HT(3A)(QDA) receptors in the presence of maximal 5-HT was best fit by three exponentials, but in the presence of ethanol a fourth open state was evident. In summary, the QDA substitution greatly enhanced single-channel conductance with little effect on 5-HT(3A) channel's kinetic properties and ethanol enhances agonist action on 5-HT(3A) receptors by inducing a new, long-lived open-channel state. Furthermore, the 5-HT(3A)(QDA) receptor appears to be suitable for pharmacological studies of 5-HT(3A) receptor modulation at a single-channel level.

BK channel subunit composition modulates molecular tolerance to ethanol.

BACKGROUND: The large conductance calcium-activated potassium channel (also called BK channel or Slo channels) is a well-studied target of alcohol action, and plays an important role in behavioral tolerance. METHODS: Using patch clamp electrophysiology, we examined human BK channels expressed in HEK293 cells to test whether tolerance to ethanol occurs in excised patches and whether it is influenced by subunit composition. Three combinations were examined: hSlo, hSlo + beta(1), and hSlo + beta(4). RESULTS: The 2 components of BK alcohol adaptation (Component 1: rapid tolerance to acute potentiation, and Component 2: a more slowly developing decrease in current density) were observed, and varied according to subunit combination. Using a 2-exposure protocol, Component 1 tolerance was evident in 2 of the 3 combinations, because it was more pronounced for hSlo and hSlo + beta(4). CONCLUSIONS: Thus, rapid tolerance in human BK occurs in cell-free membrane patches, independent of cytosolic second messengers, nucleotides or changes in free calcium. Alcohol pretreatment for 24 hours altered subsequent short-term plasticity of hSlo + beta(4) channels, suggesting a relationship between classes of tolerance. Finally, Component 2 reduction in current density showed a striking dependency on channel composition. Twenty-four hour exposure to 25 mM ethanol resulted in a down-regulation of BK current in hSlo and hSlo + beta(4) channels, but not in hSlo + beta(1) channels. The fact that hSlo + beta(1) channels show less sensitivity to acute challenge, in conjunction with less Component 1 and Component 2 tolerance, suggests subunit composition is an important factor for these elements of alcohol response.

High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.

The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA(A) receptors containing α1 and ß3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT(3A)) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [(3)H]muscimol to the purified GABA(A)R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.

Beta-subunits are important modulators of the acute response to alcohol in human BK channels.

BACKGROUND: The BK channel (a Ca2+-activated potassium ion channel encoded by the slo gene) has been defined as a target of alcohol action in a number of preparations, possibly serving as primary mediator of intoxication in the Caenorhabditis elegans model system. However, we know little of the actions of alcohol on human BK, nor the consequences of BK subunit composition on alcohol action. METHODS: Here, we use human embryonic kidney (HEK) cells to express various subunit combinations (hslo alpha+beta1 or beta4) of human BK, and examine the acute actions of alcohol on this channel using single channel recording techniques. RESULTS: The human channel is potentiated by alcohol, although the presence of the beta1, and to a lesser extent, beta4-subunit, significantly reduced acute ethanol potentiation. Potentiation increased with concentration up to an asymptote, at which point potentiation decreased. The concentration of the asymptote differed according to subunit composition. The mechanism of potentiation was also subunit-dependent, with 25 mM ethanol affecting the mean open time of hSlo+beta4 channels, whereas channel open time was unaffected by the presence of beta1. The possibility that the known effect of the beta-subunit on calcium sensitivity accounts for its modulation of acute alcohol action is discussed. CONCLUSION: Our data reinforce the idea that, as in other systems, BK may play a major role in alcohol's actions in humans, and highlight the potential role of channel subunit composition in the response to alcohol.

Bilayer thickness modulates the conductance of the BK channel in model membranes.

The conductance of the BK channel was evaluated in reconstituted bilayers made of POPE/POPS (3.3:1), or POPE/POPS with an added 20% of either SPM (3.3:1:1), CER (3.3:1:1), or CHL (3.3:1:1). The presence of SPM, which is known to increase bilayer thickness, significantly reduced the conductance of the BK channel. To directly test the role of membrane thickness, the conductance of the BK channel was measured in bilayers formed from PCs with acyl chains of increasing length (C14:1-C24:1), all in the absence of SPM. Slope conductance was maximal at a chain length of (C18:1) and much reduced for both thinner (C14:1) and thicker (C24:1) bilayers, indicating that membrane thickness alone can modify slope conductance. Further, in a simplified binary mixture of DOPE/SPM that forms a confined, phase-separated bilayer, the measured conductance of BK channels shows a clear bimodal distribution. In contrast, the addition of CER, which has an acyl chain structure similar to SPM but without its bulky polar head group to POPE/POPS, was without effect, as was the addition of CHL. The surface structure of membranes made from these same lipid mixtures was examined with AFM. Incorporation of both SPM and CER resulted in the formation of microdomains in POPE/POPS monolayers, but only SPM promoted a substantial increase in the amount of the high phase observed for the corresponding bilayers. The addition of CHL to POPE/POPS eliminated the phase separation observed in the POPE/POPS bilayer. The decrease in channel conductance observed with the incorporation of SPM into POPE/POPS membranes was, therefore, attributed to larger SPM-rich domains that appear thicker than the neighboring bilayer.