The presence and function of calcium (Ca) channels in kidney cells is poorly understood. It is known, however, that Ca channel blockers (dihydropyridines (DHP), phenylklkylamines, etc.] can affect numerous kidney cell functions, particularly in some pathophysiological states (hypertension, diabetes mellitus, ischemia, etc.). Recently, we identified a DHP-sensitive Ca channel in proximal tubules of rabbit which could be activated by swelling. The goal of the present proposal is to characterize this renal, swelling-activated, DHP-sensitive Ca channel.
Four specific aims are proposed to be achieved: 1) To characterize the functional properties of the DHP-sensitive, swelling-activated Ca channel. Using swelling-induced, DHP-sensitive Ca influx (fura-2 fluorescence) and single channel analysis (patch clamp), the Ca channel will be characterized to determine channel behavior, cell volume-dependency of activation, and DHP specificity (agonist vs antagonists). 2) To determine the signalling/second messenger pathways underlying swelling-induced activation of the DHP-sensitive Ca channel. Studies will focus on elucidating the role of likely transduction pathways (membrane depolariztion, cAMP/protein kinase A, and diacylglycerol/protein kinase C) in activating the channel during swelling. 3) To isolate and sequence a cDNA clone encoding the renal, DHP-sensitive Ca channel (alpha1-subunit) and to determine whether other subunits of DHP-sensitive Ca channels are expressed in renal cells. A short segment (408 bp) of a proximal tubule Ca channel has been cloned which is homologous to cardiac muscle DHP- sensitive, L-type Ca channels. The clone will be used to screen a proximal tubule cDNA library to obtain the full-length cDNA sequence. The expression of other Ca channel subunits in the renal cells will be assessed by Northern blot analysis. 4) To determine the functional characteristics of the cloned, DHP-sensitive Ca channel and to determine its relation to the swelling-activated, DHP-sensitive Ca channel. Based on the cloned cDNA sequence, full-length cDNA of the alpha1-subunit will be expressed (or coexpressed with cDNA of other subunits identified above) in Xenopus oocytes and the electrophysiological properties compared to those of the swelling-induced Ca channels of proximal tubule cells. Anti- sense/sense RNA methods will be utilized to modulate endogenous Ca channel expression in cultured proximal tubule cells to provide direct evidence as to whether the cloned channel is the swelling-activate channel. In summary, the project will provide a needed characterization of the functional properties, molecular structure, and mechanism of activation of an important, renal Ca channel.
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