ANION DEPENDENCE OF BUMETANIDE BINDING AND ION-TRANSPORT BY THE RABBIT PAROTID NA-K+-2CL(-) COTRANSPORTER - EVIDENCE FOR AN INTRACELLULAR ANION MODIFIER SITE()

The anion dependence of [H-3]bumetanide binding and Na-22(+) transport by the rabbit parotid Na+-K+-2Cl(-) co-transporter was studied in aci nar basolateral membrane vesicles (BLMVs). Cl-, Br- and NO3- have a bi phasic effect on binding consistent with the presence of two anion sit es associated with the bumetanide binding event, a high-affinity stimu latory site and a lower-affinity inhibitory site. We show that formate shares only the stimulatory site and SO42- only the inhibitory site. The initial rate of [H-3]bumetanide binding was stimulated by formate or low [Cl-] and inhibited by SO42- or high [Cl-], but the rate of [H- 3]bumetanide dissociation was not affected by the presence of these an ions in the dissociation medium. However, when [H-3]bumetanide was bou nd to BLMVs in the presence of formate its rate of dissociation was mo re than four times faster than when binding took place in the presence of Cl-. These observations indicate that the binding of bumetanide an d the stimulatory anion are ordered such that the anion must necessari ly bind first and subsequently cannot dissociate until after bumetanid e dissociates. In zero-trans-flux experiments, extravesicular SO42- an d formate had no effect on Na-22(+) transport via the co-transporter [ Turner and George (1988) J. Membr. Biol. 102, 71-77]. Thus neither of the anion sites associated with bumetanide binding is a Cl- transport site. However, we show here that SO42- inhibits transport when present in the intravesicular space. Since the BLMV preparation is predominan tly oriented cytosolic-side-in, this observation indicates the existen ce of an inhibitory cytosolic anion modifier site. Our data suggest th at this site is identical to the inhibitory anion site associated with bumetanide binding.