Interaction of Charybdotoxin S10A with Single Maxi-K Channels: Kinetics of Blockade Depend on the Presence of the β1 Subunit

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Abstract
The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming α subunit and a regulatory β1 subunit that modifies the biophysical and pharmacological properties of the α subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either α or α+β1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 ± 12 s, that were similar to those expressing α+β1 subunits, 51 ± 16 s. In contrast, with the α subunit alone, ChTX-S10A block times were much shorter, 5 ± 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of α+β1 subunits but not of α subunits alone. These findings indicate that it may be possible to predict the association of β1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a β1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [125I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [125I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [125I]-IbTX-D19Y/Y36F was observed into the β1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the α/β1 interaction.