Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels

Conditioning hyperpolarization-induced delays in the potassium channels of myelinated nerve.

Hyperpolarizing conditioning pulses delay the onset of potassium channel current in voltage-clamped myelinated nerve fibers. Both the development of and recovery from this conditioning are approximately exponential functions of time: the time constants are functions of the conditioning voltage. The delay is larger and develops faster for more hyperpolarized conditioning pulses. The magnitude of the delay (but not the rate of development or recovery) depends upon the test potential-small test depolarizations produce larger delays than large depolarizations. The currents with and without the conditioning pulse cannot be made to superimpose by a simple time translation

Voltage-independent gating transitions in squid axon potassium channels.

We have investigated the actions of internal and external Zn2+ on squid axon K channel ionic and gating currents. As has been noted previously, application of Zn2+ to either membrane surface substantially slowed the activation of these channels with little or no change in deactivation. Internal Zn2+ (near 200-300 nM) slowed channel activation by up to sixfold over the range of membrane voltages from -30 to +50 mV. External Zn2+ (10 mM) produced an approximate twofold slowing of activation from -40 to +40 mV. We found that the changes in ionic current activation kinetics were accompanied by less than a twofold slowing of channel-gating currents in a narrow range of potentials near -30 mV. There was, at most, only a few percent reduction of charge movement associated with Zn2+ application. We conclude that these ions interact with channel components involved in weakly voltage-dependent conformational changes. Although there are some differences in detail, the general similarity of the actions of both internal and external Zn2+ on channel function suggests that the modified channel-gating step involves amino acids accessible to both the internal and external membrane surface

Electrostatic interaction between charybdotoxin and a tetrameric mutant of Shaker K(+) channels.

The scorpion toxin, Charybdotoxin (CTX), blocks homotetrameric, voltage-gated K(+) channels by binding near the outer entrance to the pore in one of four indistinguishable orientations. We have determined the pH-dependence of CTX block of a tetrameric Shaker potassium channel with a single copy of a histidine replacing the wild-type phenylalanine at position 425. We compared this pH-dependence with that from homotetrameric channels with four copies of the mutation. We found that protonation of a single amino acid at position 425 had a large effect on the affinity of the channel for CTX-much larger than expected if only one of the four CTX binding orientations was disrupted. The pK(a) for the H(+)-ion induced protection from CTX block indicates that the electrostatic environment near position 425 is likely basic in nature, perhaps because of the proximity of lysine 427. We also examined the pH-dependence of block of channels with one and four copies of the histidine mutation by CTX containing neutralizing mutations of four basic residues on the active face of the toxin. The results suggested an orientation of CTX on the channel that places three of the positively charged CTX residues very near three of the four Shaker 425 positions