Characterization of human ASIC2a homomeric channels stably expressed in murine Ltk − cells

Abstract

ASIC2a (BNaC1 or MDEG) is distributed throughout the nervous system and potentially involved in mechanosensation, hearing, vision, and taste functions. However, pharmacological properties of ASIC2 homomers including the mechanism of inhibition by amiloride remain unclear. In this study, we describe the properties of hASIC2a stably expressed in Ltk − cells, the first reported stable cell line expressing any ASICs subunit, by standard whole cell voltage clamp method. In response to pH 4.0, at − 80 mV, hASIC2a cells exhibited rapidly activating fast transient inward current (∼ 100 pA/pF) that was followed by a sustained current (∼ 13 pA/pF). In contrast, untransfected Ltk − cells showed only a very small rapidly activating non-inactivating inward current (∼ 4 pA/pF). The magnitude of hASIC2a transient current was pH dependent with pH 50 values for activation and inactivation of ∼ 4.2 and ∼ 5.5, respectively. Ion substitution experiments revealed the following rank order of permeability: Na + > K + > Ca 2+ for the transient current. Amiloride reversibly inhibited the pH 4.0 evoked transient current with IC 50 values of ∼ 20 μM at both − 30 and − 80 mV holding potentials, indicating that the interactions are voltage independent when nearly all amiloride is protonated. Amiloride (100 μM) did not inhibit ASIC2a transient current when pre-applied in pH 7.4 and pH 4.0 currents obtained in absence of amiloride, but it did inhibit currents when co-applied at pH 4.0 suggesting open channel blockade. In summary, ASIC2a stable cell line serves as a useful model system to study the pharmacological properties of ASIC2a currents, potentially contributing to pH-evoked responses in cells of the dorsal root ganglion and the central nervous system.