Loop length matters: Effects of truncating the cytoplasmic M3M4 loop of the 5-HT3A receptors on channel function
McKinnon, Nicole K.
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The Cys-loop receptor family of neurotransmitter-gated ion channels forms receptors for acetylcholine, serotonin (5-HT), GABA and glycine. They are formed by the pentameric assembly of homologous subunits that share a common structure; with an amino-terminal extracellular domain, four transmembrane alpha-helices and a large intracellular loop (>75 residues) between M3 and M4. The extracellular domain binds agonist and contains the Cys-loop for which the superfamily is named. The second transmembrane segment lines the pore. It forms the channel gate and is a regulator of ion selectivity and conductance. The large intracellular loop contains two domains: the C-terminal membrane-associated helix (MA) and a large N-terminal region that is unstructured in the 4-A resolution acetylcholine receptor structure but is believed to be involved in receptor trafficking and synaptic targeting. The five MA helices form an inverted tepee lining the vestibule at the cytoplasmic mouth of the transmembrane channel. MA helix residues line the putative portals into the cytoplasmic vestibule and in the 5-HT3A receptor are important determinants of single channel conductance. It is unknown whether the portals also determine the size selectivity for permeant ions. Prokaryotic Cys-loop homologues lack both the extracellular Cys-loop and the large cytoplasmic loop, having instead a cytoplasmic loop of less than 15 residues. Chimeric 5-HT3A receptors with the M3M4 loop replaced by the predicted heptapeptide loop from the homologous bacterial Gloeobacter violaceus GLIC channel were functional. These findings suggested that the large cytoplasmic loop was not essential for receptor function. To date, very few studies have looked at the role of the cytoplasmic M3M4 loop on ion size selectivity, channel function and desensitization kinetics.;To examine the constraints of the M3M4 loop for receptor function we replaced the 5-HT3A M3M4 loop with one to seven alanines (5-HT 3A-An=1--7). We studied the electrophysiological and biochemical properties of the resulting channels. All mutants were functional with 5-HT EC50's similar to WT; however desensitization times differed amongst the mutants. 5-HT3A-A2, 5-HT3A-A 4, 5-HT3A-A6, or 5-HT3A-A7 had desensitization rates similar to WT; however 5-HT3A-A 3, or 5-HT3A-A5 had desensitization rates an order of magnitude faster than WT. Additionally, after the initial opening, the 5-HT3A-A1 construct entered a non-binding, non-functional state, from which it did not recover on our experimental time scale. This suggests that the large M3M4 loop of eukaryotic Cys-loop channels is not required for assembly or function; however the loop length and its amino acid composition can have significant effects on channel desensitization rates. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments in the channel protein as it undergoes gating-induced conformational changes. Thus, while the M3M4 loop is not essential for channel assembly and function it can exert significant effects on the channel kinetics.;To investigate the role of the cytoplasmic portals in ion size selectivity, we used homomeric 5-HT3A receptors with either a wild-type (WT) or chimeric heptapeptide (5-HT3-glvM3M4) M3M4 loop. The portals are absent in HT3-glvM3M4. We measured the relative conductance and permeabilities of a series of organic and inorganic cations ranging in radius from 0.9--4.5 A (Li+, Na+, ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, diethanolammonium, tetramethylammonium, choline, TRIS and NMDG). Both constructs had measurable conductances with Li+, ammonium, and methylammonium (size range 0.9--1.8 A radius). Ions larger than 2.4 A did not have measurable conductances. The permeability ratios were similar for 5-HT3A and 5-HT3-glvM3M4 receptors for all ions tested. We infer that the cytoplasmic portals, while a major determinant of conductance do not form the size selectivity filter for permeant ions.