Na+ Permeation and Block of hERG Potassium Channels

The inactivation gating of hERG channels is important for the channel function and drug–channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms

Rapid Induction of P/C-type Inactivation Is the Mechanism for Acid-induced K+ Current Inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H+o-induced current inhibition by taking advantage of Na+ permeation through inactivated channels. In hKv1.5, H+o inhibited open-state Na+ current with a similar potency to K+ current, but had little effect on the amplitude of inactivated-state Na+ current. In support of inactivation as the mechanism for the current reduction, Na+ current through noninactivating hKv1.5-R487V channels was not affected by [H+o]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H+o. These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na+ currents but the maintained presence of slow Na+ tail currents, combined with changes in the Na+ tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na+ current at low pH

Modulation of human ether-à-go-go-related K+ (HERG) channel inactivation by Cs+ and K+

Unlike many other native and cloned K+ channels, human ether-à-go-go-related K+ (HERG) channels show significant Cs+ permeability with a PCs/PK (the permeability of Cs+ relative to that of K+) of 0.36 ± 0.03 (n = 10). Here, we find that raising the concentration of external Cs+ (Cs0+) dramatically slows HERG channel inactivation without affecting activation. Replacement of 5 mm K0+ by 135 mm Cs0+ increased both inactivation and recovery time constants and shifted the mid-point of the steady-state inactivation curve by 25 mV in the depolarized direction (n = 6, P < 0.01). Raising [Cs+]o also modulated the voltage sensitivity of inactivation gating. With 130 8mm Cs1+ and 135 mm NMDGo+, the inactivation time constant decreased e-fold per 47.5 ± 1.1 mV (n = 5), and when 20 mm Cs+ was added to the bath solution, the inactivation time constant decreased e-fold per 20.6 ± 1.3 mV (n = 5, P < 0.01). A quantitative analysis suggests that Cs0+ binds to a site in the pore that is influenced by the transmembrane electrical field, so that Cs0+-induced slowing of HERG inactivation is less prominent at strong depolarizations. K0+ has effects that are similar to Cs0+ and their effects were additive, suggesting Cs0+ and K0+ may share a common mechanism of action. The strong effects of Cs+ on inactivation but not on activation highlight the importance of ion and channel interactions during the onset of inactivation in the HERG channel

The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide directly blocks human ether à-go-go-related gene potassium channels stably expressed in human embryonic kidney 293 cells

BACKGROUND AND PURPOSE N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) is a well-known calmodulin inhibitor used to study calmodulin regulation of intracellular Ca 2+ signalling-related process. Here, we have determined whether W-7 would inhibit human ether gene (hERG or K v11.1) potassium channels, hK v1.5 channels or hK IR2.1 channels expressed in human embryonic kidney (HEK) 293 cells. EXPERIMENTAL APPROACH The hERG channel current, hK v1.5 channel current or hK IR2.1 channel current was recorded with a whole-cell patch clamp technique. KEY RESULTS It was found that the calmodulin inhibitor W-7 blocked hERG, hK v1.5 and hK IR2.1 channels. W-7 decreased the hERG current (I hERG) in a concentration-dependent manner (IC 50: 3.5 μM), and the inhibition was more significant at depolarization potentials between +10 and +60 mV. The hERG mutations in the S6 region Y652A and F656V, and in the pore helix S631A, had the IC 50s of 5.5, 9.8 and 25.4 μM respectively. In addition, the compound inhibited hK v1.5 and hK IR2.1 channels with IC 50s of 6.5 and 13.4 μM respectively. CONCLUSION AND IMPLICATIONS These results indicate that the calmodulin inhibitor W-7 exerts a direct channel-blocking effect on hERG, hK v1.5 and hK IR2.1 channels stably expressed in HEK 293 cells. Caution should be taken in the interpretation of calmodulin regulation of ion channels with W-7. © 2010 The British Pharmacological Society.link_to_OA_fulltex