Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

Abstract

5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. 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 as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.

The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization

“…2A). The estimated pH value of half-maximal activation (pH 50 ) for ASIC1a was 6.32 Ϯ 0.07 (CI 6.18 -6.48), for ASIC2a was 3.54 Ϯ 0.04 (CI 3.47-3.62), for ASIC1a-F2a was 6.76 Ϯ 0.05 (CI 6.66 -6.86), for ASIC1a-T2a was 6.26 Ϯ 0.06 (CI 6.13-6.39), for ASIC1a-FT2a was 5.23 Ϯ 0.05 (CI 5.14 -5.32), for ASIC2a-T1a was 3.60 Ϯ 0.05 (CI 3.50 -3.69), and for ASIC2a-FT1a was 3.72 Ϯ 0.04 (CI 3.65-3.79) (Fig. 2B).…”

“…Assuming all null hypotheses are true, we expect 5% of the comparisons to have uncorrected p values of less than 0.05. The pH of half-maximal activation (pH 50 ) was calculated from normalized pH-evoked peak currents plotted as a function of the pH of activation. Data were fitted to a standard monotonic sigmoid dose-response curve.…”

An Internally Modulated, Thermostable, pH-sensitive Cys Loop Receptor from the Hydrothermal Vent Worm Alvinella pompejana

“…It has been shown earlier that changes in the M3-M4 loop in eukaryotic CLRs alter expression and desensitization (37)(38)(39), modulate interaction with other proteins (40), and affect the single channel conductance in cation-selective CLRs (41). Alva1-pHCl has an intracellular M3-M4 loop that is 25-30 amino acids long.…”

“…McKinnon et al (37) have shown, for the 5-HT3A receptor, that mutants with a polyalanine sequence replacing the native M3-M4 loop were functional, disfavoring a role of charged residues in loss of function. In tAlv-a1-pHCl-AGT, it may be that replacing the M3-M4 loop with the tripeptide AGT restricts the conformational flexibility of the M3 and M4 helices.…”

Characterization of the target of ivermectin, the glutamate-gated chloride channel, fromAnopheles gambiae

“…The function of this region has been shown to impact channel kinetics and trafficking in other members of the Cys-loop family and provides potential sites for post-translational modifications (Tsetlin et al, 2011;McKinnon et al, 2012). However, it is still unclear how the regions of heterogeneity between isoforms may affect channel activity and expression.…”