CL- CHANNELS IN BASOLATERAL RENAL MEDULLARY MEMBRANES XII - ANTI-RBCLC-KA ANTIBODY BLOCKS MTAL CL- CHANNELS

Cl- channels in the medullary thick ascending limb (MTAL) studied by e ither patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl- concentrations. rbClC-Ka, a ClC Cl- channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl- transportin two separat e ways. First, an antibody was raised against a fusion protein contain ing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit ki dney sections with the antibody was localized to basolateral regions o f MTAL and cortical thick ascending limb (CTAL) segments and also to t he cytoplasm of intercalated cells in the cortical collecting duct. Se cond, Cl- uptake and efflux were measured in suspensions of mouse MTAL segments. Cl- uptake was bumetanide sensitive and was stimulated by t reatment with a combination of vasopressin + forskolin + dibutyryl ade nosine 3',5-cyclic monophosphate (DBcAMP). Cl- efflux was also increas ed significantly by vasopressin + forskolin + DBcAMP from 114 +/- 20 t o 196, 36 nmol mg protein(-1).45 s(-1) (P = 0.003). Cl- efflux was inh ibited by the Cl- channel blacker diphenylamine-2-carboxylate (154 +/- 26 vs. 70 +/- 21 nmol mg protein(-1).45 s(-1), P = 0.003). An anti-rb ClC-Ka antibody, which inhibits the activity of MTAL Cl- channels in l ipid bilayers, reduced Cl- efflux from intact MTAL segments (154 +/- 2 8 vs. 53 +/- 14 nmol mg protein(-1).45 s(-1), P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl- channe l that mediates vasopressin-stimulated net Cl- transport in the MTAL s egment.