A role for the beta 1-beta 2 loop in the gating of 5-HT3 receptors

Abstract

Based on the Torpedo acetylcholine receptor structure, Unwin and colleagues (Miyazawa et al., 2003; Unwin, 2005) hypothesized that the transduction of agonist binding to channel gate opening involves a "pin-into-socket" interaction between alphaV46 at the tip of the extracellular beta1-beta2 loop and the transmembrane M2 segment and M2-M3 loop. We mutated to cysteine the aligned positions in the 5-HT3A and 5-HT3B subunit beta1-beta2 loops K81 and Q70, respectively. The maximal 5-HT-activated currents in receptors containing 5-HT3A/K81C or 5-HT3B/Q70C were markedly reduced compared with wild type. Desensitization of wild-type currents involved fast and slow components. Mutant currents desensitized with only the fast time constant. Reaction with several methanethiosulfonate reagents potentiated currents to wild-type levels, but reaction with other more rigid thiol-reactive reagents caused inhibition. Single-channel conductances of wild type, K81C, and K81C after modification were similar. We tested the proximity of K81C to the M2-M3 loop by mutating M2-M3 loop residues to cysteine in the K81C background. Disulfide bonds formed in 5-HT3A/K81C/A304C and 5-HT3A/K81C/I305C when coexpressed with 5-HT3B. We conclude that in the resting state, K81 is not in a hydrophobic pocket as suggested by the pin-into-socket hypothesis. K81 interacts with the extracellular end of M2 and plays a critical role in channel opening and in the return from fast desensitization. We suggest that during channel activation, beta1-beta2 loop movement moves M2 and the M2-M3 loop so that the M2 segments rotate/translate away from the channel axis, thereby opening the lumen. Recovery from fast desensitization requires the interaction between K81 and the extracellular end of M2.

Figure 1.

a, Homology model of a 5-HT_3A homopentameric receptor, viewed from the plane of the membrane. One subunit is highlighted in red for clarity. Green and blue regions show the β₁-β₂loop and M2-M3 loop, respectively. The dashed lines indicate the approximate limits of the lipid bilayer. b, Portion of a ClustalW multiple sequence alignment of the putative β₁-β₂ loop in several neurotransmitter-gated ion channels. The vertical arrow marks residues that align with AChR αV46, the residue proposed to be critical for the pin-into-socket mechanism for gating. The asterisks indicate absolutely conserved residues, and the dots indicate partially conserved positions. nACh, Nicotinic ACh.

Figure 2.

a, Mean peak currents recorded at a holding potential of -80 mV from 5-HT₃R containing the K81C and/or Q70C mutations, expressed in Xenopus oocytes. b, EC₅₀ values for 5-HT for WT and mutant 5-HT₃R, as in a. c, Time constants of desensitization (τ) in the presence of 5-HT (50 μm) for WT and mutant 5-HT₃R, as in a. Error bars indicate SEM; n = 6. ^*p < 0.05, significantly different from WT (ANOVA). Note that the y-axis has a log₁₀ scale in a and c.

Figure 3.

a, Representative traces showing the effect of reaction with MTSEA⁺ on currents recorded at a holding potential of -80 mV from 5-HT_3A/K81C homopentamers expressed in Xenopus oocytes. b, Sample traces showing an experiment to determine the rate of reaction of MTSEA⁺ with 5-HT_3A/K81C homopentamers expressed in Xenopus oocytes and a plot of the peak currents from this experiment. The second-order rate of reaction is calculated from the nonlinear least squares fit shown on the graph. c, Representative traces showing currents obtained on application of 5-HT to HEK293T cells expressing either WT 5-HT_3A or 5-HT_3A/K81C subunits, each along with WT 5-HT_3B subunits, and the effect of reaction with MTSET⁺. Note that in contrast to data obtained in Xenopus oocytes, desensitization could be fit by a double exponential, giving rate constants for both fast and slow desensitization.

Figure 4.

a, Modification of 5-HT_3A/K81C homopentamers by various thiol-reactive reagents irreversibly effects peak current. Currents were recorded from injected Xenopus oocytes at a holding potential of -80 mV. The responses to pairs of saturating test pulses of 5-HT (50 μm) were averaged before and after reaction with each reagent. Error bars indicate SEM; n = 3. b, Chemical structures of the products formed by reacting cysteine with each thiol-reactive reagent used in this study. The cysteinyl group is oriented similarly to the left in each case. The curved arrows denote in each case the bond with which free energy calculations were performed.

Figure 5.

Currents recorded from HEK293 cells transiently transfected with constructs coding for 5-HT_3A mutant receptors. Each trace shows the response of an outside-out patch to 50 μm 5-HT. The calibration bars refer to the full traces. The selected sections of each trace, indicated by the gray lines, are shown below at an expanded time scale. Displayed to the right of each trace is an all-points histogram of the expanded portion of the recording. QDA, The presence in all transfected subunits of the R437Q/R441D/R445A set of mutations in the intracellular M3-M4 loop, known to elevate the single-channel conductance of 5-HT_3A receptors to directly measurable levels. 5-HT_3A/WT/QDA (a), 5-HT_3A/K81C/QDA (b), and 5-HT_3A/K81C/QDA (c), after treatment of the cells for 3 min with 20 μm MTSET⁺, are shown.

Figure 6.

a, Section of a homology model of the 5-HT_3A receptor subunit, focused on the junction between the extracellular domain (top) and membrane domain (bottom). Note the predicted proximity of K81 to the extracellular portion of the M2 segment. b, Sample traces from an experiment demonstrating inhibition of the double mutant 5-HT_3A/K81C/A304C by oxidizing conditions (Cu:phen, 2 min) and relief of inhibition by reducing conditions (DTT, 3 min), indicating the reversible formation of a disulfide bond. c, Selected members of the Cys loop superfamily of ion channels aligned in the region between transmembrane segments M2 and M3. Blue letters indicate residues mutated to cysteine in this study. Underlined residues were found to cross-link with the β₁-β₂ loop in this study. Red letters indicate positions implicated previously in coupling binding to channel gating in other studies (Lynch et al., 1997; Kash et al., 2003; Sala et al., 2004). Residue numbers refer to positions from the beginning of the predicted protein precursor.