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Key points In adult ventricular myocytes, the IKs channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. Abstract Slow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There have been debates as to how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β-adrenergic signaling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulfide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. In-situ proximity ligation assay (PLA) detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein (FP)-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that in cardiomyocytes KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfill its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer. This article is protected by copyright. All rights reserved.

The hERG channel has an unusually long ‘S5–P linker’ (residues 571–613) that lines the outer mouth of the pore. Previously, we have shown that residues along this S5–P linker are critical for the fast-inactivation process and K+ selectivity of the hERG channel. Here we used several approaches to probe the structure of this S5–P linker and its interactions with other domains of the hERG channel. Circular dichroism and NMR analysis of a synthetic hERG S5–P linker peptide suggested that this linker is quite dynamic: its central region (positions 583–593) can be unstructured or helical, depending on whether it is immersed in an aqueous phase or in contact with a hydrophobic environment. Cysteine introduced into positions 583–597 of the S5–P linker can form intersubunit disulphide bonds, and at least four of them (at 584, 585, 588 and 589) can form disulphide bonds with counterparts from neighbouring subunits. We propose that the four S5–P linkers in a hERG channel can engage in dynamic conformational changes during channel gating, and interactions between S5–P linkers from neighbouring subunits contribute importantly to channel inactivation.

Mechanisms of Bupivacaine's Actions on K Channels. Introduction: We examined the effects of a nonspecific ion channel blocker, bupivacaine, on K channels encoded by hERG, rKvl.4, rKv4.3, and hKvLQTI along with hIsK. Their native counterparts in the heart are important for the function of IKr, Ito, and IKs and, thus, play an important role in repolarization. Methods and Results: To elucidate the mechanisms and sites of bupivacaine's actions, we correlated the voltage and time dependencies of drug effects with those of channel gating. We also studied the effects of altering the C-type (hERG) or N-type (rKvl.4) inactivation process or the subunit composition (hKvLQT1 with or without hIsK) on bupivacaine's actions. The results suggest that, except for hKvLQT1 co-expressed with hIsK, bupivacaine binding occurred at depolarized voltages coinciding with channel activation. With hKvLQT1 co-expressed with hIsK, bupivacaine bound preferentially at negative voltages when channels were in the closed state, and unbound at depolarized voltages when channels opened. The C-type inactivation of hERG enhanced, whereas the N-type inactivation of rKv1.4 hindered, bupivacaine's effects. Conclusion: We propose that bupivacaine's actions on these K channels can be described as a nonspecific pore blockade in the inner mouth region. However, the apparent binding affinity and voltage dependence of binding can be differentially influenced by the inactivation processes occurring at two ends of the pore (C-type inactivation at the outer end and N-type inactivation at the inner end), or by the interaction between hIsK and hKvLQT1 subunits.

Arrhythmogenic Effects of Quinidine on Catecholamine-induced Delayed Afterdepolarizations. We studied the effects of quinidine and tetrodotoxin (TTX), two drugs that block Na+ channels, on delayed afterdepolarizations (DAD) caused by norepinepbrine in atrial fibers of the canine coronary sinus. At long stimulus cycle lengths of 10 seconds, quinidine increased the amplitude of the afterdepolarizations and caused triggered activity within 1–2 minutes. Simultaneously, action potential duration (APD) was lengthened but upstroke velocity was not decreased. Prolonged exposure to quinidine eventually decreased upstroke velocity but DAD amplitude remained larger than control and triggered activity was still induced more easily. The effects of quinidine to increase afterdepolarizations was partially related to its prolongation of the APD since shortening APD with repolarizing current decreased DAD amplitude. However, DAD amplitude remained larger than control indicating that quinidine caused triggering by other mechanisms as well. TTX, on the other hand, which blocks Na+ channels but shortens APD, decreased DAD amplitude and triggered activity. Part but not all of these effects resulted from the shortening of APD by TTX since prolongation of APD with depolarizing current only partially restored DAD amplitude. Anti-arrhythmic drugs, therefore, may have effects on DADs that partly result from changes in the APD. Quinidine may cause cardiac arrhythmias by virtue of its effects to potentiate triggered activity.

The human ether-a-go-go-related gene (hERG) encodes the pore-forming component of the rapid delayed rectifier (IKr) channel that helps maintain the electrical stability of the heart. A key feature of hERG/IKr gating is its fast inactivation process, which is important for its physiological function. All available data suggest that hERG/IKr 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 Erev for high-K+ selectivity (e.g. wild-type hERG, Erev ≈−100 mV in 2 mm [K]o−96 mm [Na]o) and Erev 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 PK:PNa) in the hERG mutants is not an ‘all-or-none’ phenomenon, but instead is graded. Therefore, it is important to measure the shift in Erev by removing extracellular Na+ ions. For less K+-selective channels, removing Nao will shift Erev in the negative direction. This allows a quantitative comparison of PK:PNa 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 Erev (e.g. −70 mV) is due to a marginal reduction in PK:PNa, or a loss of [K]i in unhealthy oocytes. Therefore, we routinely quantified PK:PNa 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 PK:PNa 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/IKr channels.