Novel isoforms of the beta and gamma 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 alpha, beta, and gamma 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 beta 2 and gamma 2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta 2 and gamma 2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate, The physiological and pharmacological profile of the Na channel expressed after alpha beta 2 gamma XENaC cRNA injection in Xenopus oocyte did not differ from alpha beta gamma XENaC. By contrast, the channel expressed after alpha beta gamma 2 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 alpha beta gamma 2; 1.34 mu M versus alpha beta gamma 0.35 mu M). These data indicate that the gamma (and/or gamma 2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration, The close homology between gamma and gamma 2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.