Biophys J, July 1998, p. 218-225, Vol. 75, No. 1

A Distinct Contribution of the  Subunit to Acetylcholine Receptor Channel Activation Revealed by Mutations of the M2 Segment

Department of Biophysical Sciences, State University of New York at Buffalo, Buffalo, New York 14214 USA

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (, , , and ), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with  subunits having a binding site mutation that slows channel opening (D200N). In these AChRs, a 12'P mutation in  or  slowed the closing rate constant ~6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (K_d). In contrast, a 12'P mutation in  slowed the channel closing rate constant only ~2-fold and significantly increased both the channel opening rate constant and the K_d. Pairwise expression of 12'P subunits indicates that mutations in  and  act nearly independently, but one in  reduces the effect of a homologous mutation in  or . The results suggest that a 12'P mutation in  and  has mainly local effects, whereas one in  has both local and distributed effects that influence both agonist binding and channel gating.

Biophys J, July 1998, p. 218-225, Vol. 75, No. 1
© 1998 by the Biophysical Society   0006-3495/98/07/218/08  $2.00

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