Voltage-dependent block by zinc of single calcium channels in mouse myotubes

1. The blocking actions of Zn2+ on currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. 2. Adding 100 microM-Zn2+ to the patch electrode containing 110 mM-BaCl2 produced an increase in the open channel noise, presumably arising from unresolved blocking and unblocking of the open channel by Zn2+. Adding between 200 and 1000 microM-Zn2+ to the electrode reduced the amplitude of the unitary current in a concentration-dependent manner. 3. The single-channel current-voltage (i-V) relations showed that Zn2+ reduced the amplitude of the unitary Ba2+ currents at all potentials more negative than 0 mV. A plot of the amplitude of the unitary current in the presence of Zn2+, normalized to the amplitude in its absence, showed that block of the current depended on voltage, decreasing as the patch potential was made more negative. 4. The normalized amplitudes of the unitary currents were plotted as a function of the logarithm of [Zn2+] in the electrode. The relation for currents recorded at different potentials were fitted to an expression for binding to a single site with a KD at 0 mV of approximately 500 microM. The KD changed approximately e-fold per 83 mV with hyperpolarization. The results suggest Zn2+ binds to a site located at approximately 15% of the potential drop from the surface membrane. 5. Reducing the concentration of Ba2+ in the patch electrode enhanced the steady-state block of unitary currents by Zn2+. The inverse of the unitary current was plotted as a function of [Ba2+]o in the presence and absence of Zn2+; both were linear and intersected at the ordinate, indicating Ba2+ and Zn2+ compete for a channel site. 6. The kinetics of Zn2+ block of unitary Ba2+ currents were studied by amplitude distribution analysis. As expected for a simple reaction between blocking ion and open channel, the blocking rate depended linearly on the concentration of Zn2+, while the exit rate was independent of concentration. The second-order rate coefficient for Zn2+ entry in the presence of 110 mM-BaCl2 at 0 mV was approximately 2.0 X 10(7) M-1S-1, while the exit rate was approximately 16000 s-1. 7. Both entry and exit rates increased as the membrane potential was made more negative. The entry rate increased approximately e-fold per 66 mV, while the exit rate increased approximately e-fold per 41 mV.(ABSTRACT TRUNCATED AT 400 WORDS)