22 G. D. Cymes and C. Grosman
to become slower when deuterium oxide replaces water as the solvent because
of the known effect of isotopic substitution on the properties of the ground states
and transition state of acid–base reactions (Bell 1973 ). In fact, in recent work
on the muscle AChR in which we studied the open-channel current fluctuations
caused by mutations to the ring of (naturally occurring) glutamates in the charge-
selectivity filter, it was precisely the lack of pH dependence and the insensitiv-
ity to isotopic substitution that prompted us to identify a completely different
mechanism for this phenomenon (Cymes and Grosman 2012 ; Harpole and Gros-
man 2014 ).
3 Concluding Remarks
Thus far, the single-channel approach presented here has only been applied to the
muscle AChR. Although it is not a foregone conclusion that individual proton-
ation and deprotonation events manifest electrophysiologically in other channels
as clearly as they do in the muscle AChR, we certainly hope that the method we
described here will be successfully applied to other systems, as well. A relatively
high single-channel conductance and the possibility of mutating only a single
residue per channel seem to be a good starting point. Also, we think that in ad-
dition to providing structural information about well-defined functional states of
a protein in its membrane environment, the method can be used to gain valuable
insight into the electrostatics of ion-channel pores. Specifically, we envision that
this approach can be applied to test such experimentally elusive ideas in ion-
channel biophysics as the hypothesis of hydrophobic gating and to provide ex-
perimental benchmarks to computational approaches aimed at predicting protein
side-chain pKa values.
References
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