Isolation and reconstitution of <Ca^(2+)> -dependent <K^+> channel

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Apamin is a neurotoxic peptide present in the venom of honey bee. When applied to the sympathetic ganglion cells of bullfrog, apamin did not affect either the <Ca^(2+)> channel or the <Ca^(2+)> -dependent <K^+> channel involved in the falling phase of the action potential. Apamin specifically blocked the <Ca^(2+)> -dependent <K^+> channel that genarates the afterhyperpolarization. Voltage clamp studies showed that the <Ca^(2+)> -dependent <K^+> current consists of at least 2 components responsible for the afterhyperpolarization; fast decaying component and slowly decaying component. The time constant of the two components were about 50 and 250 msec. Apamin decreased the time constant of the former without changing its amplitude and decreased the amplitude of the latter without changing its time constant. These results indicate that apamin is a specific probe for certain types of <Ca^(2+)> -dependent <K^+> channel.Synaptic plasma membrane (SPM) prepared from rat brain was incubated with <^(125)I> -apamin and then bifunctional cross-linker was added to cross-linke the apamin-receptor complex. Autoradiographs of two-dimensional gels gave a single acidic protein of <M_r> about 30,000. The density of the apamin receptor was of the order of 10 fmol/mg protein, too low to be purified by ordinary biochemical means. We extracted the radiolabeled spot from the gel and concentrated it to immunize a BALB/c mouse to obtain polyclonal and monoclonal antibodies to the receptor. However, it has not been successful so far.