The anion specificity of the sodium-potassium-chloride cotransporter in rabbit kidney outer medulla: studies on medullary plasma membranes

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Abstract
Plasma membrane vesicles were isolated from rabbit kidney outer medulla and employed in sodium, rubidium, and chloride flux studies. Chloride dependence and bumetanide sensitivity of (part of) the sodium and rubidium flux indicate that this plasma membrane fraction can be used to study the properties of Na−K−2Cl cotransport system present in the luminal membrane of the medullary thick ascending limb. The anion specificity of the cotransporter was investigated by determining the effect of anion replacement on sodium fluxes. When chloride was completely replaced by bromide, iodide, nitrate, or thiocyanate only bromide could effectively substitute for chloride (90% activity), whereas sodium uptake in the presence of iodide, nitrate, and thiocyanate amounted to only 25% of the sodium uptake observed in the presence of chloride. When similar replacement experiments were performed in the presence of 10 mmol/l chloride, bromide could substitute for chloride by 110%, iodide and nitrate by 60%, and thiocyanate by 70%. In the presence of 10 mmol/l bromide iodide, nitrate, and thiocyanate were similarly effective. The effect of nitrate and chloride on sodium flux was additive. Bumetanide-sensitive chloride uptake was inhibited by nitrate, the inhibition was however only partly, amounting to 60%. The results obtained are compatible with the view that the two anion binding sites of the Na−K−2Cl cotransporter can exhibit a different substrate specificity and that the transporter in addition to a 2Cl mode can also operate in a 2Br, Cl−, A− and Br−, A− mode, A− representing iodide, nitrate, or thiocyanate.

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