Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.
Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.
Anderson
C S
CS
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
MacKinnon
R
R
Smith
C
C
Miller
C
C
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D018999:Charybdotoxin; D004553:Electric Conductivity; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism; D008051:Lipid Bilayers; D008564:Membrane Potentials; D009132:Muscles / Q000378:metabolism; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D012604:Scorpion Venoms / Q000494:pharmacology*
D007473:Ion Channels; D008051:Lipid Bilayers; D012604:Scorpion Venoms; D018999:Charybdotoxin; D011188:Potassium; D002118:Calcium