Saturable high-affinity binding of [3H]bumetanide [dissociation constant (K(D)) = 80 nM] was measured in microsomal membranes prepared from squid optic ganglia. Under control conditions, the maximal specific binding of labeled bumetanide (B(max)) was ~6-7 pmol/mg protein. Binding had a higher relative affinity for bumetanide than for furosemide and dependend on the presence of Cl- and K+, but not Na+, in the incubation media. In the case of K+, [3H]-bumetanide binding was half-saturated at [K+] = 100 mM. The Cl- effect was biphasic. At [Cl-] between 0 and 150 mM, [3H]-bumetanide binding increased with increasing [Cl-]. However, when [Cl-] was increased above 150 mM, [3H]bumetanide binding was progressively reduced. ATP acted as a nonessential activator [mean affinity constant (K0.5) ~1 μM] of the ion-dependent [3H]bumetanide binding by increasing the apparent binding capacity. The activation by ATP did not require Mg2+. Other adenosine analogues also stimulated the binding of bumetanide.
- adenosine 5'-trisphosphate
- loop diuretics
- sodium-potassium-chloride cotransport
ASJC Scopus subject areas
- Cell Biology