Abstract
The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na(+) with Li(+), K(+), Rb(+), or Cs(+) altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P(K)/P(Na) = 0.1 and G(K)/G(Na) = 0.7 and P(Rb)/P(Na) = 0.03 and G(Rb)/G(Na) = 0.3. Stimulation of Cl(-) currents when Na(+) was replaced with Ca(2+), Sr(2+), or Ba(2+) indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na(+) in a hyperbolic manner, consistent with an apparent affinity (K(m)) for Na(+) conduction of 25 mM. Nitrogen-containing cations, including NH4(+), NH3OH(+), and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na(+) currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na(+) (K(m) = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.