A cytoplasmic region determines single-channel conductance in 5-HT3 receptors

Abstract

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.

Dynamic Modification of a Mutant Cytoplasmic Cysteine Residue Modulates the Conductance of the Human 5-HT3A Receptor

“…The transient application of 5-HT (10 M) to outside-out membrane patches clamped at Ϫ74 mV elicited rapidly rising inward currents from which unitary events with a mean ␥ of 7.8 Ϯ 0.5 pS (Table 1) were discernable during current deactivation ( Table 1). Since the ␥ of WT 5-HT 3A receptors is too low to enable direct observation of unitary events (3,4,8), we used homomeric mutant 5-HT 3A (R436D) receptors as a control to exclude potential effects of MTSEA that may occur independently of the substituted cysteine residue. MTSEA had no significant effect on the ␥ of events mediated by homomeric 5-HT 3A (R436D) receptors, which was 9.1 Ϯ 0.6 and 9.8 Ϯ 0.9 pS in the absence and presence of the reagent, respectively (Table 1).…”

“…MTS Reagents Modulate ␥ of the Mutant 5-HT 3A (R432Q,R436C,R440A) Receptor-In subsequent experiments, we employed a 5-HT 3A (R432Q,R436C,R440A) construct, hereafter termed 5-HT 3A (QCA), which we anticipated from our previous studies (3,4) would produce channels with a ␥ more amenable to the quantitative analysis of the modulatory effects of MTS reagents. In comparison with 5-HT 3A (R436C), the ␥ of the 5-HT 3A (QCA) construct determined from recordings performed on outside-out membrane patches was indeed substantially increased (17.7 Ϯ 0.4 pS) ( Fig.…”

“…Although the role of residues within the TM2 helix of Cys loop receptors in controlling ␥ is well established, recent studies demonstrate a similar function for residues located in the large cytoplasmic TM3-4 loop (1,3,4). Based on a 4.6 Å resolution cryoelectron microscopy image of the Torpedo marmorata nACh receptor, Miyazawa et al (5) first speculated that amino acids in the TM3-4 loop form "transverse tunnels" that may contribute to the ion conduction pathway.…”

“…Based on a 4.6 Å resolution cryoelectron microscopy image of the Torpedo marmorata nACh receptor, Miyazawa et al (5) first speculated that amino acids in the TM3-4 loop form "transverse tunnels" that may contribute to the ion conduction pathway. Subsequent electrophysiological studies demonstrated that cytoplasmic residues within membrane-associated (MA) helices of the 5-HT 3 and ␣ 4 ␤ 2 nACh receptors are determinants of ␥ (3,4). Arg 436 in the 5-HT 3A subunit at a position termed MA 0Ј is critical for maintaining the anomalously low ␥ (ϳ900 femtosiemens) that is a hallmark of the homomeric 5-HT 3A receptor.…”

“…Point mutations were introduced into the 5-HT 3A construct using standard molecular biological techniques, and all constructs were sequenced to confirm fidelity. Transient transfection of HEK293 or tsA-201 cells with cDNA was performed using the calcium phosphate precipitation method or electroporation, respectively, as described previously (3,8). Cells were subcultured twice weekly and incubated in a medium composed of Dulbecco's modified Eagle's medium and 10% calf serum, supplemented with 100 g/ml streptomycin and 100 units/ml penicillin.…”