Subunit composition determines the single channel kinetics of the epithelial sodium channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of alpha with either wild-type beta or gamma subunits and alpha with carboxy-terminal truncated beta (beta(T)) or gamma (gamma(T)) subunits in Xenopus laevis oocytes. alpha beta and alpha beta(T) channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 mu M amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P-o) for alpha beta was 0.54 and for alpha beta(T) was 0.50. In comparison, alpha gamma and alpha gamma(T) channels exhibited different kinetics: alpha gamma channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high P-o (0.78), or short openings with long closed times, resulting in a low P-o (0.16). The mean P-o for all alpha gamma channels was 0.48. alpha gamma(T) (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P-o of 0.6, similar to that of alpha gamma. Inclusion of 0.1 mu M amiloride in the pipette solution reduced the mean open time of alpha gamma(T) to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of alpha beta, alpha beta(Upsilon) (1 mu M amiloride), and alpha gamma(T) (0.1 mu M amiloride) channels. alpha beta and alpha beta(T) had similar blocking and unblocking rate constants, whereas the unblocking rate constant for alpha gamma(T) was 10-fold slower than alpha beta(T). Our results indicate that subunit composition of ENaC is a main determinant of P-o. In addition, channel kinetics and P-o are not altered by carboxy-terminal deletion in the beta subunit, whereas a similar deletion in the gamma subunit affects channel kinetics but not P-o.