Linkage between ‘disruption of inactivation’ and ‘reduction of K⁺ selectivity’ among hERG mutants in the S5-P linker region

The human ether-á-go-go-related gene (hERG) encodes the pore-forming component of the rapid delayed rectifier (I_Kr) channel that helps maintain the electrical stability of the heart. A key feature of hERG/I_Kr gating is its fast inactivation process, which is important for its physiological function. All available data suggest that hERG/I_Kr inactivation is similar to the P-type inactivation described for the Shaker and some other Kv channels: conformational changes around the selectivity filter leading to an obstruction of current flow through the pore. However, what is unique in the hERG channel is its unusually long S5-P linker, that is critically involved in the inactivation process. How this occurs is of great interest to experimentalists and protein modellers, and is being pursued by several laboratories including mine.

With this background, I would like to comment on an article recently published in The Journal of Physiology by Clarke et al. (2006). In the Discussion (p. 301, right column), the authors stated ‘…. the S5P α-helix charge scan mutants did not show significant shifts in reversal potential…… This is in contrast to the majority of the S5P α-helix cysteine that had marked changes in reversal potential (Liu et al. 2002). It has subsequently been shown that the majority of these S5P α-helix cysteine mutants form intersubunit disulphide bonds (Jiang et al. 2005) and thus have significantly disrupted outer pore structure.’ In essence, Clarke et al. implied that data interpretation in Liu et al. (2002) was incorrect.

As the senior author on both papers cited by Clarke et al. I would like to point out that in the Liu et al. (2002) article we carried out the essential experiments to rule out the possibility that the reduced K⁺ selectivity observed in some of the cysteine mutants (16 out of 51) was due to disulphide bridge formation. We used DTT treatment and an increase in channel sensitivity to thiol-modifying methanethiosulphonate (MTS) reagents to monitor the progression of disulphide bond reduction. Cysteine mutants in the disulphide bonded state (before DTT treatment) had little or no MTS sensitivity. DTT treatment broke disulphide bonds and increased the MTS sensitivity (Figs 3 and 4 of Liu et al. 2002). With this test, we clearly showed that although in many of the cysteine mutants DTT treatment increased MTS sensitivity and restored high K⁺ selectivity, in the others DTT treatment increased MTS sensitivity without changing the low K⁺ selectivity (Fig. 6 of Liu et al. 2002). It is worth noting that even if DTT treatment reduced disulphide bonds in only a fraction of the channels, if these reduced channels manifested a high K⁺ selectivity, we would have picked up the signal by closely monitoring the tail current morphology at different repolarization potentials. That is, in the voltage range between E_rev for high-K⁺ selectivity (e.g. wild-type hERG, E_rev ≈−100 mV in 2 mm [K]_o−96 mm [Na]_o) and E_rev for low-K⁺ selectivity (−20 to −70 mV), we would have observed biphasic tail currents with an initial inward component (originating from the low-K⁺ selectivity population) superimposed on a slower outward component (originating from the high-K⁺ selectivity population). In fact, such biphasic tail currents were observed during progression of disulphide bond reduction in those cysteine mutants that eventually restored a high K⁺ selectivity when the reduction was complete. The inescapable explanation from these experiments was that some of the cysteine mutants had reduced K⁺ selectivity even when their introduced thiol groups were in reduced state, i.e. the loss of K⁺ selectivity in these mutant channels was not due to disulphide bond formation and a global deformation of the outer mouth. We called these ‘high-impact’ positions, to acknowledge the fact that these positions could not tolerate cysteine substitution while many other positions could.

Note that the reduction in K⁺ selectivity (reduction in the K⁺ to Na⁺ permeability ratio or P_K:P_Na) in the hERG mutants is not an ‘all-or-none’ phenomenon, but instead is graded. Therefore, it is important to measure the shift in E_rev by removing extracellular Na⁺ ions. For less K⁺-selective channels, removing Na_o will shift E_rev in the negative direction. This allows a quantitative comparison of P_K:P_Na values among mutant channels, and is needed for correctly concluding whether K⁺ selectivity is altered or not. Otherwise, it will be difficult to conclude whether a somewhat less negative E_rev (e.g. −70 mV) is due to a marginal reduction in P_K:P_Na, or a loss of [K]_i in unhealthy oocytes. Therefore, we routinely quantified P_K:P_Na for mutant hERG channels in our studies (Fan et al. 1999; Dun et al. 1999; Liu et al. 2002).

It has long been recognized that P-type inactivation in Kv channels is accompanied by an increase in Na⁺ permeability: Shaker (Starkus et al. 1997), Kv2.1 (Korn & Ikeda, 1995), Kv1.5 (Wang et al. 2000), and very recently hERG (Gang & Zhang, 2006). Therefore, it is not surprising that mutations in the S5-P linker that perturb hERG's ability to inactivate can also affect its ability to discriminate between Na⁺ and K⁺ ions. Structurally, this suggests that certain residues in hERG's S5-P linker intimately interact with the channel's selectivity filter.

Referring back to the observations by Clarke et al. that charge mutations at four positions in the hERG's S5-P linker could disrupt inactivation without altering the pore's K⁺ selectivity, it is important to distinguish between two ‘inactivation–disruption’ mutant phenotypes: due to ‘a marked positive shift in the voltage dependence of inactivation’ versus‘inability to inactivate’. The mutants described by Clarke et al. belong to the first phenotype. There is a well-known precedent: the S631A mutation of hERG disrupts inactivation by shifting the voltage dependence of inactivation by approximately +100 mV. However, hERG-S631A can still inactivate at strongly depolarized voltages and maintains a high K⁺ selectivity. On the other hand, in the cysteine mutants at the high-impact positions we studied, there was no inactivation no matter how depolarized the membrane voltage was. This belongs to the second phenotype, and is accompanied by a loss of K⁺ selectivity. It is possible that the two distinct phenotypes are caused by different mechanisms and have different structural bases. In summary, I would like to emphasize that, when studying mutational effects on hERG inactivation and K⁺ selectivity, it is important to quantify graded changes in P_K:P_Na and define how mutations disrupt inactivation. A careful and thorough quantification of mutational effects on these channel properties is prerequisite to identifying the mechanism of hERG inactivation, a subject important for the physiology, pharmacology and pathology of hERG/I_Kr channels.