Extracellular Protons Inhibit Charge Immobilization in the Cardiac Voltage-Gated Sodium Channel

AbstractLow pH depolarizes the voltage-dependence of cardiac voltage-gated sodium (NaV1.5) channel activation and fast inactivation and destabilizes the fast-inactivated state. The molecular basis for these changes in protein behavior has not been reported. We hypothesized that changes in the kinetics of voltage sensor movement may destabilize the fast-inactivated state in NaV1.5. To test this idea, we recorded NaV1.5 gating currents in Xenopus oocytes using a cut-open voltage-clamp with extracellular solution titrated to either pH 7.4 or pH 6.0. Reducing extracellular pH significantly depolarized the voltage-dependence of both the QON/V and QOFF/V curves, and reduced the total charge immobilized during depolarization. We conclude that destabilized fast-inactivation and reduced charge immobilization in NaV1.5 at low pH are functionally related effects

K(+) Activation of Kir3.1/Kir3.4 and Kv1.4 K(+) Channels Is Regulated by Extracellular Charges

K(+) activates many inward rectifier and voltage-gated K(+) channels. In each case, an increase in K(+) current through the channel can occur despite a reduced driving force. We have investigated the molecular mechanism of K(+) activation of the inward rectifier K(+) channel, Kir3.1/Kir3.4, and the voltage-gated K(+) channel, Kv1.4. In the Kir3.1/Kir3.4 channel, mutation of an extracellular arginine residue, R155, in the Kir3.4 subunit markedly reduced K(+) activation of the channel. The same mutation also abolished Mg(2+) block of the channel. Mutation of the equivalent residue in Kv1.4 (K532) abolished K(+) activation as well as C-type inactivation of the Kv1.4 channel. Thus, whereas C-type inactivation is a collapse of the selectivity filter, K(+) activation could be an opening of the selectivity filter. K(+) activation of the Kv1.4 channel was enhanced by acidic pH. Mutation of an extracellular histidine residue, H508, that mediates the inhibitory effect of protons on Kv1.4 current, abolished both K(+) activation and the enhancement of K(+) activation at acidic pH. These results suggest that the extracellular positive charges in both the Kir3.1/Kir3.4 and the Kv1.4 channels act as “guards” and regulate access of K(+) to the selectivity filter and, thus, the open probability of the selectivity filter. Furthermore, these data suggest that, at acidic pH, protonation of H508 inhibits current through the Kv1.4 channel by decreasing K(+) access to the selectivity filter, thus favoring the collapse of the selectivity filter

Base of Pore Loop Is Important for Rectification, Activation, Permeation, and Block of Kir3.1/Kir3.4

The Kir3.1/Kir3.4 channel is an inward rectifier, agonist-activated K(+) channel. The location of the binding site within the channel pore that coordinates polyamines (and is thus responsible for inward rectification) and the location of the gate that opens the channel in response to agonist activation is unclear. In this study, we show, not surprisingly, that mutation of residues at the base of the selectivity filter in the pore loop and second transmembrane domain weakens Cs(+) block and decreases selectivity (as measured by Rb(+) and spermine permeation). However, unexpectedly, the mutations also weaken inward rectification and abolish agonist activation of the channel. In the wild-type channel and 34 mutant channels, there are significant (p < 0.05) correlations among the K(D) for Cs(+) block, Rb(+) and spermine permeation, inward rectification, and agonist activation. The significance of these findings is discussed. One possible conclusion is that the selectivity filter is responsible for inward rectification and agonist activation as well as permeation and block

Base of Pore Loop Is Important for Rectification, Activation, Permeation, and Block of Kir3.1/Kir3.4

The Kir3.1/Kir3.4 channel is an inward rectifier, agonist-activated K(+) channel. The location of the binding site within the channel pore that coordinates polyamines (and is thus responsible for inward rectification) and the location of the gate that opens the channel in response to agonist activation is unclear. In this study, we show, not surprisingly, that mutation of residues at the base of the selectivity filter in the pore loop and second transmembrane domain weakens Cs(+) block and decreases selectivity (as measured by Rb(+) and spermine permeation). However, unexpectedly, the mutations also weaken inward rectification and abolish agonist activation of the channel. In the wild-type channel and 34 mutant channels, there are significant (p < 0.05) correlations among the K(D) for Cs(+) block, Rb(+) and spermine permeation, inward rectification, and agonist activation. The significance of these findings is discussed. One possible conclusion is that the selectivity filter is responsible for inward rectification and agonist activation as well as permeation and block