Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small

Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block

A constriction formed by the crossing of the second transmembrane domains of ASIC1, residues G432 to G436, forms the narrowest segment of the pore in the crystal structure of chicken ASIC1, presumably in the desensitized state, suggesting that it constitutes the “desensitization gate” and the “selectivity filter.” Residues Gly-432 and Asp-433 occlude the pore, preventing the passage of ions from the extracellular side. Here, we examined the role of Asp-433 and Gly-432 in channel kinetics, ion selectivity, conductance, and Ca2+ block in lamprey ASIC1 that is a channel with little intrinsic desensitization in the pH range of maximal activity, pH 7.0. The results show that the duration of open times depends on residue 433, with Asp supporting the longest openings followed by Glu, Gln, or Asn, whereas other residues keep the channel closed. This is consistent with residue Asp-433 forming the pore’s closing gate and the properties of the side chain either stabilizing (hydrophobic amino acids) or destabilizing (Asp) the gate. The data also show residue 432 influencing the duration of openings, but here only Gly and Ala support long openings, whereas all other residues keep channels closed. The negative charge of Asp-433 was not required for block of the open pore by Ca2+ or for determining ion selectivity and unitary conductance. We conclude that the conserved residue Asp-433 forms the closing gate of the pore and thereby determines the duration of individual openings while desensitization, defined as the permanent closure of all or a fraction of channels by the continual presence of H+, modulates the on or off position of the closing gate. The latter effect depends on less conserved regions of the channel, such as TM1 and the extracellular domain. The constriction made by Asp-433 and Gly-432 does not select for ions in the open conformation, implying that the closing gate and selectivity filter are separate structural elements in the ion pathway of ASIC1. The results also predict a significantly different conformation of TM2 in the open state that relieves the constriction made by TM2, allowing the passage of ions unimpeded by the side chain of Asp-433