Eldefrawi_1978_Biochemistry_17_5474

The electric organ of the ray, Torpedo ocellata, can serve as a source for both the acetylcholine (ACh) receptor and its ionic channel. The two entities were identified by their specific binding of [3H]ACh and [3H]perhydrohistrionicotoxin ([3H]H12-HTX), respectively. Binding of [3H]H12-HTX was inhibited by certain drugs and toxins, e.g., histrionicotoxin (HTX), amantadine, and tetraethylammonium (TEA) ions at concentrations that did not inhibit [3H]ACh binding. However, the specific carbamoylcholine-induced 22Na efflux from microsacs from the electric organ membranes was blocked by inhibitors of either the receptor or its ionic channel. The ionic channel had the properties of a protein as judged by heat sensitivity and the inhibition of [3H]H12-HTX binding, after incubation of the electric organ membranes with protein reagents such as p-chloromercuribenzenesulfonic acid (PCMBS) or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The "binding" of [3H]H12-HTX at 4 X 10(-8) M to lipids in the microsacs was 12% of the total binding to intact microsacs and was nonsaturable and insensitive to heat or specific drugs. After solubilization with cholate, the [3H]H12-HTX binding subunits retained the same affinities for toxins and drugs. The Kd for [3H]H12-HTX was 3 X 10(-7) M. The majority of the ionic channel could be separated from the ACh receptors in the cholate extract by incubation with ACh-receptor affinity gel and ACh-receptor antibodies. The ACh receptor purified by this affinity gel contained only a few active ionic channel units as judged by low levels of high affinity binding of [3H]H12-HTX. On the other hand, after solubilization with Triton X-100, all the ionic channel molecules were either separated or denatured so that the purified ACh receptor did not exhibit high affinity binding for [3H]H12-HTX.