Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit.
The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha 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 alpha or alpha+beta 1 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 alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha 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 alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 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 beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-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 [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 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 alpha/beta 1 interaction.
Giangiacomo
K M
KM
Department of Biochemistry, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA. giang@unix.temple.edu
Fremont
V
V
Mullmann
T J
TJ
Hanner
M
M
Cox
R H
RH
Garcia
M L
ML
D000818:Animals; D001011:Aorta; D002417:Cattle; D018999:Charybdotoxin / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D006801:Humans; D007700:Kinetics; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D024681:Potassium Channels, Calcium-Activated; D011485:Protein Binding; D011817:Rabbits; D055672:Static Electricity
D003432:Cross-Linking Reagents; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D018999:Charybdotoxin; C064719:iberiotoxin