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Original Article |
A Single-Channel Study of Second Transmembrane Segment 12' Mutants
Department of Physiology and Biophysics, School of Medicine and Biomedical Sciences, SUNY at Buffalo, 124 Sherman Hall, Buffalo, NY, 14214.716-829-2569
grosman{at}buffalo.edu
The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12' position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C
O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the 
12' residue makes a more pronounced contribution to unliganded gating than the homologous residues of the 
, β, and 
subunits; (c) combinations of second transmembrane segment 12' mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at 12' is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (Y93F, Y190W, and D200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12' mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.
Key Words: nicotinic receptors • allosteric proteins • site-directed mutagenesis
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