Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

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Abstract
In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4′-dibenzamido-2,2′-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, τDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50,DBDS,MCD (0.5±0.1 μm) for the H2-DIDS (4,4′-diisothiocyano-2,2′-dihydrostilbene disulfonate) inhibition of τDBDS is in agreement with the ID50,Cl −,MCD (0.94±0.07 μm) for H2-DIDS inhibition of MCD cell Cl− flux, thus relating τDBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl− exchange by a factor of two, from τCl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 μm, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (K I,K +,rbc=0.017 μm). These experiments indicate that the Na+,K−-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076±0.005 μm); 2 μm CE also more than doubles the Cl− exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

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