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Inhibition of voltage-dependent calcium channels by omega-conotoxin MVIIC (omega-CTx-MVIIC) was studied in various types of rat neurons. When studied with 5 mM Ba2+ as charge carrier, omega-CTx-MVIIC block of N-type calcium channels in sympathetic neurons was potent, with half-block at 18 nM. Block of N-type channels had a rapid onset (tau approximately 1 sec at 1 microM omega-CTx-MVIIC) and quick reversibility (tau approximately 30 sec). The rate of block was proportional to toxin concentration, consistent with 1:1 binding of toxin to channels, with a rate constant (k on) of approximately 1 X 10(6) M-1. sec-1. Both potency and rate of block were reduced dramatically with increasing concentrations of extracellular Ba2+ omega-CTx-MVIIC also blocked P-type calcium channels in cerebellar Purkinje neurons, but both development and reversal of block were far slower than for N-type channels. The rate of block was proportional to toxin concentration, with k on -1.5 x 10(3) M-1. sec-1 at 5 mM Ba2+. From this value and an unblocking time constant of approximately 200 min, a dissociation constant of approximately 50 nM was estimated. Thus, block of P-type channels is potent but very slow. In hippocampal CA3 pyramidal neurons, omega-CTx-MVIIC blocked approximately 50% of the high-threshold calcium channel current; one component (approximately 20%) was blocked with the rapid kinetics expected for N-type channels, whereas the other component was blocked slowly. The component blocked slowly was reduced but not eliminated by preexposure to 200 nM or 1 microM omega-Aga-IVA.

Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2-5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low-threshold T-type current, the resistant current had rapidly decaying tail currents.

We investigated whether the cysteine residues in Shaker potassium (K+) channels are essential for activation and inactivation gating or for modulation of activation gating by external zinc (Zn2+). Mutants of the Shaker K+ channel were prepared in which all seven cysteine residues were replaced (C-less). These changes were made in both wild-type Shaker H4 channels and in a deletion mutant (delta 6-46) lacking N-type ("fast") inactivation. Replacement of all cysteines left most functional properties of the K+ currents unaltered. The most noticeable difference between the C-less and wild-type currents was the faster C-type inactivation of the C-less channel which could be attributed largely to the mutation of Cys462. This is consistent with the effects of previously reported mutations of nearby residues in the S6 region. There were also small changes in the activation gating of C-less currents. Modulation by external Zn2+ of the voltage dependence and rate of activation gating is preserved in the C-less channels, indicating that none of the cysteines in the Shaker K+ channel plays an important role in Zn2+ modulation.