Leu85 in the beta1-beta2 linker of ASIC1 slows activation and decreases the apparent proton affinity by stabilizing a closed conformation

Abstract

Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The beta1-beta2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor.

FIGURE 1.

Properties of Xenopus xASIC1.1 and xASIC1.2 and effect of mutations in the β1-β2 linker in proton affinity. A, cladogram generated by Clustal alignment of ASIC proteins. Xen, Xenopus. B, whole-cell currents of xASIC1.1 and xASIC1.2 evoked by sequential application of solutions of pH 7.3, 7.1, 6.8, and 6.5. Between stimuli, the external solution was returned to pH 8.0 for 30 s. C, dose response curves to protons of xASIC1.1 (X.1), xASIC1.2 (X.2), and rat ASIC1a (Rat). D, amino acid sequence alignment of transmembrane domain 1 and the β1-β2 linker of rat, Xenopus, and EFS ASIC1. E, dose response curves to protons of xASIC1.1 (WT) with a pH_50D of 7.8, n = 10, pH_50A of 7.0 (n = 3.2), and the mutant xASIC1-P83S-K84Q-M85L (SQL) with a pH_50D of 7.35 (n = 9.4) and a pH_50A of 7.1 (n = 3.7). Symbols represent the mean of 8–10 independent cells ± S.D. Lines are the fit of the data to Equation 1. Error bars are ± standard deviation (S.D.).

FIGURE 2.

Time course of the current of Xenopus ASIC1.1 wild type and mutants. A, normalized whole-cell currents of wild type Xenopus ASIC1.1 (blue) and rat ASIC1a (green). Currents were evoked by pH 6.8 in the presence of 120 mm Na⁺ in the bath and a holding potential −60 mV. B, normalized currents of xASIC1.1 wild type (WT) and mutant channels bearing single or three mutations in the β1-β2 linker evoked by a change in pH from 8.0 to 7.1. C, same current traces as in B but the time scale is expanded 10-fold. Bars below traces indicate the time scale.

FIGURE 3.

Patch clamp recordings of xASIC1 and mutant xASIC1-SQL. A, currents from wild type xASIC1.1 evoked by the indicated concentrations of protons recorded from a patch in the outside configuration. Protocol was as follows: 4 s of pH 8.0 followed by 8 s of activating pH. The arrow marks the initiation of stimulus. The patch contains ∼400 channels. B, currents from xASIC1.1-SQL. Protocol was as follows: 8 s of pH 8.0 and 12 s activating pH. C, currents from patches containing xASIC1 channels activated by low concentrations of protons to observe single channel events. At pH 7.3, individual openings are evident. D, currents from patches containing xASIC1-SQL. WT, wild type.

FIGURE 4.

The β1-β2 linker stabilizes a closed conformation of ASIC1. A, scheme of the simplest mechanism of ASIC1, where C is the closed nonprotonated state, C_H represents the ensemble of different conformations the channel undergoes as it binds protons prior to opening. At least three protons are needed to open the channel, and nine protons are needed for steady-state desensitization according to the Hill coefficients of obtained in Fig. 1C. O_H is the open protonated state, and D_H is the desensitized state. The rate constants are denoted by the symbols on the arrows, and k₋₁/k₊₁ is the equilibrium constant for the initial binding step of protons. B and C, histograms of open times of wild type and mutant SQL xASIC1.1. The lines are fits of the data to single exponentials. D, rising phase of averaged current responses from patches with wild type (WT) or SQL xASIC channels. Currents are scaled to the same peak current value. The region of the onset curve >20% of maximum is well described by a single exponential function. This part of the curve is determined primarily by the channel opening rate rather than agonist binding so that the time constant of the exponential function approximates the reciprocal of the opening rate. Onset times were as follows: 2 ms at pH 6.5, 1.5 ms at pH 6.0, and 1.4 ms at pH 5.0.

FIGURE 5.

Elephant shark ASIC1. A, wild type (wt) EFS ASIC1 produces small and fast currents. B, substitutions of residues in the β1-β2 linker for the corresponding SQL of rat results in large currents. C, comparison of normalized currents of wild type and SQL mutant EFS channels. Red lines are fits of the currents to Equation 2. Wild type versus SQL mutant: τ_a 3 versus 16 ms, and τ_d 17 versus 140 ms. E, apparent proton affinities of EFS-SQL. E, Western blot of surface biotinylated proteins from 15 oocytes expressing the corresponding channels. The blot was probed with a V5 antibody.