The effect of stretch on the cation permeability properties of ENaC in planar lipid bilayers

A low conductance, amiloride-sensitive Na+ channel has been cloned from rat distal colon by Canessa al. (Nature (1994) 367:463-467). This channel, termed rENaC, consists of three homologous subunits, a, , and y, and forms single Naselective channels when incorporated into planar lipid bilayers. rENaC can be activated following imposition of a hydrostatic pressure gradient (AP) across the bilayer membrane. When AP = 0, the relative cation permeability sequence (determined from reversal potential measurements made under biionic conditions) of rENaC was: PL|(1.72) > PN>(1.00) > PK(0.11) > > PRb (0.01) - PC, (<0.01). Moreover, rENaC was inhibited by either Ca!(K/ = 74 ±12 M) or Mg2 (K,1 = 6.5 ±0.8 mM). When AP = 0.26 mm, single channel open probability increased from 0.15 ±0.03 to 0.65 ± 0.04, and rENaC lost its ability to discriminate between monovalent and divalent cations. Its relative permeability sequence under maximally stretched conditions was: PLi (1.12) > PN, (1.00) > PK (0.71) PMg (0.62) > Pc, (0.54) > PRb (0.32) > PC, (0.15) PS, (0.01) = PB, (<O.OI). Buffering the bilayer bathing solution with 10 mM EGTA (i.e., [Ca2]frtt < 1 nM) resulted in activation of a, , >-rENaC (P0 = 0.65 ±0.06) when AP = 0. Imposition of a hydrostatic pressure gradient under Ca2+-free conditions did not further activate these channels. These results suggest that activation rENaC produced by membrane stretch may be due to release of divalent cation block.