Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor.

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.

Evidence for a centrally located gate in the pore of a serotonin-gated ion channel.

Serotonin-gated ion channels (5-HT3) are members of the ligand-gated channel family, which includes channels that are opened directly by the neurotransmitter acetylcholine, GABA, glycine, or glutamate. Although there is general agreement that the second transmembrane domain (M2) lines the pore, the position of the gate in the M2 is less certain. Here, we used substituted cysteine accessibility method (SCAM) to provide new evidence for a centrally located gate that moves during channel activation. In the closed state, three cysteine substitutions, located on the extracellular side of M2, were modified by methanethiosulfonate (MTS) reagents. In contrast, 13 cysteine substitutions were modified in the open state with MTS reagents. The pattern of inhibition (every three to four substitutions) was consistent with an alpha helical structure for the middle and cytoplasmic segments of the M2 transmembrane domain. Unexpectedly, open-state modification of two amino acids in the center of M2 with three different MTS reagents prevented channels from fully closing in the absence of neurotransmitter. Our results are consistent with a model in which the central region of the M2 transmembrane domain is inaccessible in the closed state and moves during channel activation.