Novel isoforms of the β and γ subunits of the Xenopus epithelial Na channel provide information about the amiloride binding site and extracellular sodium sensing

We have previously identified three homologous subunits α, β, and γ of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed β2 and γ2, which share 90 and 92% sequence identity with the previously characterized β and γ XENaC, respectively. β2 and γ2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after αβ2γ XENaC cRNA injection in Xenopus oocyte did not differ from αβγ XENaC. By contrast, the channel expressed after αβγ2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (K(I) αβγ2; 1.34 μM versus αβγ 0.35 μM). These data indicate that the γ (and/or γ2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between γ and γ2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.