Structural Differences in the Nicotinic Acetylcholine Receptor (NACHR) ion Channel Between Open and Desensitized States Revealed by Time-Resolved Photolabeling with [ 3H]Chlorpromazine (CPZ)

Abstract

Extensive photoaffinity labeling studies and mutational analyses of Torpedo/muscle-type nAChR indicate only subtle differences in the structure of the ion channel between closed, open and desensitized states, with amino acids on the same face of each M2 helix lining the ion channel in each state (cytoplasmic to synaptic: M2-2, M2-6, M2-9, M2-13, M2-16/17, & M2-20). To provide further information about the differences in channel structure between the open and desensitized states, we have used time-resolved photolabeling to determine where the channel blocker [3H]CPZ binds in the open channel state after 30 msec exposure to [3H]CPZ and agonist, compared to its binding site(s) in nAChRs in the desensitized state exposed to [3H]CPZ for 30 msec or at equilibrium. In the desensitized state, at equilibrium [3H]CPZ binds to and photolabels a pocket near the cytoplasmic end of the channel (bottom, M2-2/M2-6/M2-9) and a pocket near the extracellular end of the channel (top, M2-16/17/M2-20), while in the closed, resting state it photolabels only the site at the bottom of the channel [Chiara,DC, et.al. Biochemistry 2009 48:10066-77]. For nAChRs pre-equilibrated with agonist (desensitized state), after 30 msec [3H]CPZ photolabels only the site at the top of the channel (M2-16/17/20), while for nAChRs in the open state (30 msec agonist and CPZ), the pockets at both the bottom (M2-2/M2-6/M2-9) and top (M2-16/17/M2-20) were photolabeled. From these results we conclude that: 1) a structural alteration at the top of the ion channel occurs between closed and open states, whereas this region is similar in structure in the open and desensitized states; and 2) in the equilibrium desensitized state, there is an access barrier for CPZ at or near M2-13 (Supported by GM58448).