Structure of the M2 Transmembrane Segment of GLIC, a Prokaryotic Cys Loop Receptor Homologue from Gloeobacter violaceus, Probed by Substituted Cysteine Accessibility

Rishi B Parikh,Moez Bali,Myles H Akabas

doi:10.1074/jbc.m111.221895

Abstract

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Highlights

Channel pore is formed along the radial axis and is lined by the five M2 segments, one from each subunit (6 –10)
Multiple experimental approaches are necessary to gain a clearer view of structure/function relationship in functional GLIC channels
At pH Ͻ 5.0, we show that the GLIC channels desensitized on a time scale of minutes and the rate of desensitization increased with decreasing pH (Fig. 1A)

Summary

Introduction

Channel pore is formed along the radial axis and is lined by the five M2 segments, one from each subunit (6 –10). PCMBS modification significantly increased the subsequent currents elicited by low pH test pulses for both mutants (Fig. 2C, purple residues). Second Order Rate Constants for Cysteine Modification— Measuring the pCMBS reaction rate with the engineered Cys mutants provides information about the relative reactivity of different residues and of the reactivity of the same residue in different functional states, i.e. pH 7.5 (resting) versus 5.0 (submaximally activated).

Results

Conclusion