Binding of bumetanide to microsomes from optic ganglia of the squid, Loligo pealei

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 (K_0.5) ~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.