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.
Highlights
Three identical or homologous subunits associate to form functional channels [4]
In general, species with high plasma pH have lower temperature, lower partial pressure of CO2, and lower sodium concentration than mammals [6], all of which change the degree of ionization of water and of charged groups in proteins, it remains to be shown whether the compound effect of those factors is sufficient to render the ASIC1 response to equivalent levels across species
We found that ASIC1s from these species have higher proton affinities than the mammalian channel and that residues underlying the differences in proton affinities are in the 1-2 linker of the extracellular domain rather than in the proton sensor
Summary
Dept. of Cellular and Molecular Physiology, Yale University, 333 Cedar St., New Haven, CT 065208026. Membrane potential was held at Ϫ60 mV, and whole-cell currents were recorded with a clamp OC-725B (Warner Instrument Corp., Hamden, CT) and digitized at a sampling rate of 2 kHz (PowerLab 4/30, ADInstruments). Elephant shark Time constants for the rise and decay phases were obtained by ASIC was cloned by RT-PCR from a brain cDNA library pro- fitting the time course of currents to the function (Equation 2), vided by Dr Byrappa Venkatesh Biotinylation of Proteins and Western Blotting—Fifteen tions were as follows: 120 mM NaCl, 2 mM KCl, 1.5 mM CaCl2, oocytes per conditions were biotinylated with sulfo-NHS-SS- and 15 mM HEPES-MES titrated from pH 8.0 to 5.0. Affinity for activation (pH50A) was measured by preconditioning cells with pH 8.0 followed by stimuli of pH 7.2 to 5.5.
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