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

Summary

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, τ_DBDS, 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 ID_50,DBDS,MCD (0.5±0.1 μm) for the H₂-DIDS (4,4′-diisothiocyano-2,2′-dihydrostilbene disulfonate) inhibition of τ_DBDS is in agreement with the ID_50,Cl ⁻,_MCD (0.94±0.07 μm) for H₂-DIDS inhibition of MCD cell Cl⁻ flux, thus relating τ_DBDS 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 τ_Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,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 ID_50,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.