POINT MUTATIONS IN ALPHA-BENAC REGULATE CHANNEL GATING, ION SELECTIVITY, AND SENSITIVITY TO AMILORIDE

We have generated two site-directed mutants, K504E and K515E, in the c u subunit of an amiloride-sensitive bovine epithelial Na+ channel, alp ha bENaC. The region in which these mutations lie is in the large extr acellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepare d from Xenopus oocytes expressing either K504E or K515E alpha bENaC ar e incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are ai l altered as compared to the wild-type protein. The mutated channels e xhibit either a reduction or a complete lack of its characteristic bur st-type behavior, significantly reduced Na+:K+ selectivity, and an app roximately 10-fold decrease in the apparent inhibitory equilibrium dis sociation constant (K-i) for amiloride. Single-channel conductance for Na+ was not affected by either mutation, On the other hand, both K504 E and K515E alpha bENaC mutants were significantly more permeable to K +, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as bein g important to the regulation of fundamental channel properties.