Characterization of a partially degraded Na+ channel from urinary tract epithelium

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Abstract
The mammalian urinary bladder contains in its apical membrane and cytoplasmic vesicles, a cation-selective channel or activating fragment which seems to partition between the apical membrane and the luminal (or vesicular space). To determine whether it is an activating fragment or whole channel, we first demonstrate that solution known to contain this moiety can be concentrated and when added back to the bladder causes a conductance increase, with a percent recovery of 139±25%. Next, we show that using tip-dip bilayer techniques (at 21°C) and a patch-clamp recorder, the addition of concentrated solution resulted in the appearance of discrete current shots, consistent with the incorporation of a channel (as opposed to an activating fragment) into the bilayer. The residency time of the channel in the bilayer was best described by the sum of two exponentials, suggesting that the appearance of the channel involves an association of the channel with the membrane before insertion. The channel is cation selective and more conductive to K+ than Na+ (by a factor of 1.6). It has a linearI–V relationship, but a singlechannel conductance that saturates as KCl concentration is raised. This saturation is best described by the Michaelis-Menten equation with aK m of 160mm KCl and aG max of 20 pS. The kinetics of the channel are complex, showing at least two open and two closed states. Since the characteristics of this channel are similar to a channel produced by the degradation of amiloride-sensitive Na+ channels by the proteolytic enzyme kallikrein (which is released by the cortical collecting duct of the kidney), we suggest that this channel then is not synthesized by the cell but is rather a degraded form of the epithelial Na+ channel.

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