Block of Current through Single Calcium Channels by Fe, Co, and Ni Location of the Transition Metal Binding in the Pore Site

The blocking actions of Fe 2÷, Co 2÷, and Ni 2÷ on unitary currents carried by Ba 2÷ through single dihydropyridine-sensitive Ca 2÷ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCI z produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarizat ion speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni ~÷ differs from that produced by Fe 2÷ and Co 2+ because Ni 2÷ enters the channel ~ 20 times more slowly and exits ~ 50 times more slowly. Ni 2÷ also differs from the other transition metals because at millimolar concentrations it reduces the ampli tude of the unitary current in a concentra t ion-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size. I N T R O D U C T I O N Cat ions o f the t ransi t ion meta l series b ind to specific sites on pro te ins and are i m p o r t a n t in the catalytic activity o f many enzymes. Al though these ions genera l ly b ind at sites dist inct f rom the Ca 2+ b ind ing sites of prote ins , they share the c o m m o n feature o f blocking vo l tage-ga ted Ca 2÷ currents (reviewed in Hagiwara and Byerly, 1981; Edwards, 1982). T h e t ransi t ion metals have been used extensively to block vo l tage-ga ted Ca 2+ currents in a variety of cells and, in some instances, to separa te individual componen t s o f Ca 2÷ channel cur ren t (reviewed in Bean, 1989). While it has been assumed these ions block cur ren t flow by b ind ing to a site within the channel , Address reprint requests to Dr. J. B. Lansman, Department of Pharmacology, School of Medicine, University of California, San Francisco, CA 94143-0450. Dr. Kelly's present address is University Laboratory of Physiology, South Parks Road, Oxford, UK. J. GEN. PHVSEOC. © The Rockefeller University Press • 0022-1295/91/02/0351/17 $2.00 351 Volume 97 February 1991 351-367 on July 2, 2017 jgp.rress.org D ow nladed fom 352 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 97. 1991 there is little d i rec t informat ion on the mechan i sm by which these ions inhibi t the flow of p e r m e a n t ions. T h e p resen t study was unde r t a ke n with this goal in mind. We invest igated the block of Ba z+ currents th rough single d ihydropyr id ine-sens i t ive Ca z÷ channels in mouse myotubes by the g roup VIII t rans i t ion meta l ions Fe z+, Co z÷, and Ni z÷. T h e results show that, when observed at the s ingle-channel level, the block p r o d u c e d by Fe z+ and Co z+ differed fundamenta l ly f rom that p r o d u c e d by Ni z+ even though thei r s teady-state affinities for the channe l fall within a similar range . T h e results a re discussed in terms o f the physical p rope r t i e s o f these ions that d e t e r m i n e the rates of b locking and unblock ing and possible s t ructural features of the p o r e revealed by the b locking kinetics. A pre l iminary r e p o r t o f this work has been pub l i shed as an abstract (Winegar et al., 1990).