Structure of the M2 transmembrane segment of GLIC, a prokaryotic Cys loop receptor homologue from Gloeobacter violaceus, probed by substituted cysteine accessibility

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.

FIGURE 1.

pH response and pCMBS effects on wild type GLIC and C26L GLIC. A, application of low pH pulses to oocytes expressing wild type GLIC as indicated by the black bars above the traces. Channels desensitize on the minutes time scale with exposure to pH < 5.0. The perfusion buffer pH is 7.5 unless otherwise indicated. B, consecutive application of pH 5.0 and 200 μm pCMBS for 2 min to an oocyte expressing wild type GLIC. C, application of low pH pulses to oocytes expressing the cysteine-free C26L GLIC. Note that the pH activation of C26L GLIC is shifted to lower pH compared with the wild type. D, consecutive application of low pH and 200 μm pCMBS for 2 min to an oocyte expressing C26L GLIC. The zero current level is indicated by the dashed line. The holding potentials were −60 mV.

FIGURE 2.

The location of Cys mutants in the GLIC channel structure using 3EAM Protein Data Bank coordinates. A, location of endogenous Cys-26 in the GLIC extracellular domain. View from the GLIC channel, four subunits have been removed. The subunit is light purple, Cys-26 is red in the space-filling format, the β1 strand is cyan, the β8-β9 loop and β9 strand are yellow, and the M2 segment is blue. B, 90° rotation of the view in A. The color scheme is the same as in A. C, view of the membrane-spanning, channel-lining region showing the positions of the nonfunctional Cys mutants. Residues for which the Cys mutants were nonfunctional are shown on the middle subunit in space-filling format. Cyan, residues for which no function was elicited. Purple, 13′ and 15′ residues that were functional following pCMBS modification. To visualize the channel-lining residues, the front two subunits have been removed. The remaining three subunits are color-coded to distinguish them in this and in all subsequent panels in this figure. The middle subunit is colored light purple, with the M2 and M2-M3 loop regions that were mutated to Cys in this study in blue. The two flanking subunits are colored light green, except for the region studied, which is dark green. Note that the Cys mutants were present in all subunits but are only shown in one to simplify the figure. D, close-up view of the extracellular end of M2 to illustrate the position of the pCMBS reactive residues in space-filling format. Orange, pCMBS modification potentiated subsequent currents. Yellow, pCMBS modification inhibited subsequent currents. Red, 10′ residue where pCMBS modified gating kinetics. Cyan, nonfunctional mutants. Blue, Cys mutants not affected by pCMBS. E, view of the M2 segments looking into the channel. All of the residues are shown in space-filling format. The extracellular domain of the subunit on the right is shown in ribbon format to clarify the extent of that domain in the other subunits. Note that based on the crystal structure, many of the pCMBS-reactive residues are buried in the protein interior. F, view of the lipid bilayer facing side of the same three subunits seen in E. This view results from a 180° rotation around the vertical axis of E. The color scheme is the same as in D. Note that only one of the pCMBS reactive residues, Pro-249 (26′), has significant accessibility from the external surface of the protein. G, same view as in F, except that the M1, M3, and M4 segments have been removed from the light purple subunit to reveal the “buried” pCMBS-reactive residues on the non-channel-facing side of the M2 segment.

FIGURE 3.

Average proton-induced currents at pH 5.0 for the constructs used. Black bars indicate currents that were significantly different from water-injected oocytes by one-way analysis of variance with Tukey's post hoc test. White bars indicate currents that were not significantly different from water-injected oocytes. *, L231C (8′) was constitutively conductive at pH 7.5. Note that the x axis has a log scale.

FIGURE 4.

Irreversible effects of a 2-min application of 200 μm pCMBS on proton-induced currents. A, application of pCMBS at pH 7.5. B, application of pCMBS at pH 5.0. Black bars indicate mutants in which the average ratio of the proton-induced currents following pCMBS exposure to those before exposure was significantly different from the average ratio in C26L GLIC by one-way analysis of variance with Tukey's post test. Application of pCMBS to oocytes expressing A233C (10′) did not significantly alter the magnitude of the proton-induced current, but the response was altered as shown in Fig. 5. The effect of pCMBS application at pH 7.5 or 5.0 was not determined in oocytes expressing L231C (8′) or I232C (9′), and the effect of pCMBS application at pH 5.0 was not determined in oocytes expressing wild type GLIC. Note that the x axis has a log scale.

FIGURE 5.

Application of 200 μm pCMBS to oocytes expressing A233C (10′) for 2 min at pH 7. 5 (A) and pH 5.0 (B). The oocytes were continuously perfused with pH 7.5 buffer except as indicated by the black bars above the current traces. The zero current level is indicated by the dashed line. The holding potentials were −60 mV.

FIGURE 6.

Measurement of pCMBS reaction rates. A, example of a position where the modification by pCMBS inhibits the pH-induced current. Currents from an oocyte expressing K247C elicited by alternating 2-min applications of pH 5.0 test pulses and 0.2 μm pCMBS in pH 7.5 buffer as indicated by the black bars over the current traces. B, the pH 5.0 induced currents from A were normalized to the initial current and plotted as a function of cumulative concentration × duration of pCMBS exposure. The solid line is the single exponential fit of the data. The calculated second order reaction rate constant is 103,000 ± 3200 m⁻¹ s⁻¹. C, example of a position where the modification by pCMBS potentiates the pH-induced current. Currents from an illustrative oocyte expressing the F237C mutant elicited by alternating 2-min applications of pH 5.0 test pulses and 2 or 20 μm pCMBS in pH 7.5 buffer as indicated by the black bars over the current traces. D, the pH 5.0 currents from C were normalized by the initial current, plotted as a function of the cumulative concentration duration of pCMBS exposure, and fit to the shown equation describing the kinetics of two consecutive pseudo-first order reactions. The calculated reaction rates were 4,600 ± 220 m⁻¹ s⁻¹. E, example of a measurement of the rate of reaction of pCMBS in the activated state induced by applying pCMBS at pH 5.0. The currents shown are from an oocyte expressing the F237C mutant. The reaction rate was obtained by fitting the current change following pCMBS application with a single exponential decay function. Application of 0.1 μm pCMBS at pH 5.0 is indicated by the black bar above the current traces. The portion of the current trace during pCMBS exposure was fit with a single exponential function (indicated by the circles) to calculate the reaction rate constant of 89,000 ± 29 m⁻¹ s⁻¹.

FIGURE 7.

Second order rate constants for pCMBS modification of reactive residues. We could not fit the data for modification at 25′ (T248C) to either kinetic model. Note that the x axis has a log scale.

FIGURE 8.

The pH dependence of the second order rate constant for reaction between pCMBS and free cysteine. The experimental data points (dots) are the measured second order rate constants. Each data point is the average of at least eight reactions. The error bars are smaller than the data points. The reaction proceeded too rapidly to measure at pH > 6.5. The curve is a 1/Y²-weighted nonlinear regression of the data with Equation 2 using GraphPad Prism version 5.02 for Windows. Because we expect the error in our data to be relative to the magnitude, this weighting is applicable. Note that the y axis has a log scale.

SCHEME 1

FIGURE 9.

Normalized reactivity of cysteine mutants to pCMBS. The second order rate constants reported in Fig. 7 have been normalized to the second order rate constant for the reaction between pCMBS and free cysteine at pH 7.5 or pH 5.0. Note that the x axis has a log scale.