Our goals are to characterize basolateral C1- channels from medullary thick ascending limbs of Henle (mTAL). Antidiuretic hormone (ADH) increases NaC1 absorption in microperfused mouse mTAL segments. One exploration for this effect is that ADH changes the stoichiometry of apical salt entry from Na: C1 to Na:K:2C1; and ADH increases the functional number of apical K+ channels. ADH also augments basolateral C1 conductance in mouse mTAL segments. One proposed mechanism for this effect is that ADH, via the adenylate cyclase cascade, may directly affect basolateral C1 channels analogs to cAMP effects on C1 channel activity of apical membranes in trachea and small intestine. We have proposed that increases in mTAL C1 concentrations may activate basolateral C1 channels directly, so that intracellular C1 -the substrate for basolateral C1 channels- may be the key determinant of basolateral C1-channel activity. In this regard, work in our laboratory on single C1-channels fused into planar bilayers for basolaterally-enriched vesicles prepared either from rabbit outer medulla or from highly enriched (97%) suspensions of mouse mTAL segments indicates that the open time probability Po in such channels was augmented by increasing progressively the KC1 concentrations facing the intracellular surfaces on these channels form 2 mM to 50 mM (within the physiologic range of intracellular mTAL C1-concentrations). The catalytic subunit of protein kinase A plus ATP (C-PKA + ATP) augmented Po when 2 mM KC1 bathed intracellular faces of these C1- channels, but (c-PKA + ATP) had no effect on Po when 50 mM KC1 bathed intracellular C1- channel faces. Thus so long as intracellular mTAL C1- concentrations are not depleted, cAMP may be a biologically redundant modifier of basolateral mTAL C1-channels. These finding relating Po, (cAMP + ATP) and KC1 concentrations bathing intracellular C1-channel faces are unique among epithelial C1-channels, and may account for the fact that, in vivo, the mTAL is spared in cystic fibrosis. Our key goals are: 1) To verify identify between C1- channels in bilayers and basolateral C1-channels of intact mTAL cells; ii) To define further the biophysical properties of these C1-channels; iii) to purify basolateral mTAL C1-channels to and to reconstitute activity of purified channels in liposomes; iv) To clone the cDNA for basolateral C1-channels of the mTAL.
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