To identify the residues exposed in the channel lumen, we applied the substitutedcysteine-accessibility method to the first two membrane-spanning segments (M1 and M2) and the M1-M2 loop in two subunits, a and p. Consecutive residues were mutated one at a time to cysteine, and the mutants were expressed in oocytes. Of 107 mutants, 105 yielded functional receptors. We tested the susceptibility of the mutants to irreversible alteration of ACh-induced current by extracellularly applied methanethiosulfonate (MTS) reagents, including MTSethylammonium (MTSEA) and MTSethyltrimethylammonium (MTSET). Assuming that only SH at the wateraccessible surface of the receptor will react with these reagents and that the only water-accessible surface in the membrane-spanning domain is the lining of the channel, we identified about 25 residues that line the channel. Many residues reacted at different rates in the open and closed states of the channel, reflecting widespread conformational changes in M1 and M2. Also, in the open state, but not in the closed state, the open-channel blocker QX314 protected residues towards the cytoplasmic end of the channel against reaction. Five mutants, extending from aT244 at the cytoplasmic end of M2 into the M1-M2 loop were expressed in HEK cells, which were patch-clamped in the whole-cell configuration. In the closed state of the channel, extracellular MTSEA and the slightly larger MTSET reacted only with ocT244C. Intracellular MTSEA and MTSET reacted with aG240C and <xE241C. In the open state, extracellular MTSEA reacted with ocT244C, aK242C, aE241C, and aG240C. The position of the gate, therefore, is between aE241 and aT244. The voltage-dependence of the rate of reaction of aT244C in the open state implies that its electrical distance from the extracellular side is about 0.4; thus, as much as 0.6 of the membrane potential difference falls across the open gate. Supported by NINDS, MDA, and the McKnight Endowment Fund for Neuroscience.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology