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Inherited cardiac arrhythmias contribute substantially to sudden cardiac death in the young. The underlying pathophysiology remains incompletely understood because of the lack of representative study models and the labour-intensive nature of electrophysiological patch clamp experiments. Whereas patch clamp is still considered the gold standard for investigating electrical properties in a cell, optical mapping of voltage and calcium transients has paved the way for high-throughput studies. Moreover, the development of human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) has enabled the study of patient specific cell lines capturing the full genomic background. Nevertheless, hiPSC-CMs do not fully address the complex interactions between various cell types in the heart. Studies using in vivo models, are therefore necessary. Given the analogies between the human and zebrafish cardiovascular system, zebrafish has emerged as a cost-efficient model for arrhythmogenic diseases. In this review, we describe how hiPSC-CM and zebrafish are employed as models to study primary electrical disorders. We provide an overview of the contemporary electrophysiological phenotyping tools and discuss in more depth the different strategies available for optical mapping. We consider the current advantages and disadvantages of both hiPSC-CM and zebrafish as a model and optical mapping as phenotyping tool and propose strategies for further improvement. Overall, the combination of experimental readouts at cellular (hiPSC-CM) and whole organ (zebrafish) level can raise our understanding of the complexity of inherited cardiac arrhythmia disorders to the next level.

Voltage-gated K + (Kv) channels mediate the flow of K + across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K + over Na + selectivity, and even NMDG + can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist beta-alanine, we observed a release of IL-6. beta-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the G alpha q/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-kappa B) and stimulation of IL-6 release. This IL-6 release could be blocked by a G alpha q inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for beta-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the I-K1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of I-K1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as I-K1. By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately -20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased.

In their Letter (1), Pisupati et al. comment that they found a different Kv2.1:Kv6.4 stoichiometry (2) than we report in Moller et al. (3). Our manuscript was still under review when their article was published. Realizing that the results were inconsistent, we added an objective evaluation algorithm calculating relative probabilities for the different stoichiometry models (3). The authors of the Letter (1) speculate that our lower probability of fluorescence ( p f), an intrinsic property of the green fluorescent protein (GFP), is due to prebleaching or too fast bleaching rate. We can exclude both possibilities. We handle our preparations exclusively under low light with a wavelength of >600 nm. After focusing with lower light … [↵][1]1To whom correspondence may be addressed. Email: rikard.blunck{at}umontreal.ca. [1]: #xref-corresp-1-1

In the Na(v)channel family the lipophilic drugs/toxins binding sites and the presence of fenestrations in the channel pore wall are well defined and categorized. No such classification exists in the much larger K(v)channel family, although certain lipophilic compounds seem to deviate from binding to well-known hydrophilic binding sites. By mapping different compound binding sites onto 3D structures of Kv channels, there appear to be three distinct lipid-exposed binding sites preserved in K(v)channels: the front and back side of the pore domain, and S2-S3/S3-S4 clefts. One or a combination of these sites is most likely the orthologous equivalent of neurotoxin site 5 in Na(v)channels. This review describes the different lipophilic binding sites and location of pore wall fenestrations within the K(v)channel family and compares it to the knowledge of Na(v)channels.

Heterotetramer voltage-gated K+ (KV) channels KV2.1/KV6.4 display a gating charge-voltage (QV) distribution composed by two separate components. We use state dependent chemical accessibility to cysteines substituted in either KV2.1 or KV6.4 to assess the voltage sensor movements of each subunit. By comparing the voltage dependences of chemical modification and gating charge displacement, here we show that each gating charge component corresponds to a specific subunit forming the heterotetramer. The voltage sensors from KV6.4 subunits move at more negative potentials than the voltage sensors belonging to KV2.1 subunits. These results indicate that the voltage sensors from the tetrameric channels move independently. In addition, our data shows that 75% of the total charge is attributed to KV2.1, while 25% to KV6.4. Thus, the most parsimonious model for KV2.1/KV6.4 channels’ stoichiometry is 3:1.

Voltage-dependent K(+) channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6(T)) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6(T). Exchanging both L45 and S6(T) transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6(T) in hKv1.5 further shows that S6(T) needs to be alpha-helical and forms a "crevice" in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6(T) crevice, whereas other residues in S6(T) and L45 that are not involved in the interaction maintain the correct structure of the coupling.

The Kv1–4 families of K+ channels contain a tandem proline motif (P XP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial “fast” mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered P XP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the P XP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the P XP motif may serve as a “hinge” and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them “reluctant” to open subsequently.

Enhancement of neuronal M-currents, generated through KV7.2-KV7.5 channels, has gained much interest for its potential in developing treatments for hyperexcitability-related disorders such as epilepsy. Retigabine, a KV7 channel opener, has proven to be an effective anticonvulsant and has recently also gained attention due to its neuroprotective properties. In the present study, we found that the auxiliary KCNE2 subunit reduced the KV7.2-KV7.3 retigabine sensitivity approximately 5-fold. In addition, using both mammalian expression systems and cultured hippocampal neurons we determined that low μM retigabine concentrations had ‘off-target’ effects on KV2.1 channels which have recently been implicated in apoptosis. Clinical retigabine concentrations (0.3–3 μM) inhibited KV2.1 channel function upon prolonged exposure. The suppression of the KV2.1 conductance was only partially reversible. Our results identified KV2.1 as a new molecular target for retigabine, thus giving a potential explanation for retigabine’s neuroprotective properties.

Alkanols are small aliphatic compounds that inhibit voltage-gated K+ (Kv) channels through a yet unresolved gating mechanism. Kv channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker Kv channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type Kv channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state.

High-frequency action potential (AP) transmission is essential for rapid information processing in the central nervous system. Voltage-dependent Kv3 channels play an important role in this process thanks to their high activation threshold and fast closure kinetics, which reduce the neuron's refractory period. However, premature Kv3 channel closure leads to incomplete membrane repolarization, preventing sustainable AP propagation. Here, we demonstrate that Kv3.1b channels solve this problem by producing resurgent K+ currents during repolarization, thus ensuring enough repolarizing power to terminate each AP. Unlike previously described resurgent Na+ and K+ currents, Kv3.1b's resurgent current does not originate from recovery of channel block or inactivation but results from a unique combination of steep voltage-dependent gating kinetics and ultra-fast voltage-sensor relaxation. These distinct properties are readily transferrable onto an orthologue Kv channel by transplanting the voltage-sensor's S3–S4 loop, providing molecular insights into the mechanism by which Kv3 channels contribute to high-frequency AP transmission., Kv3 potassium channels have an important role in the repolarization of action potentials in fast-spiking neurons. Here, the authors use electrophysiology and modelling to report on an interesting mechanism that might explain their gating behaviour.

The voltage−gated K+ (Kv) channel subunits Kv2.1 and Kv2.2 are expressed in almost every tissue. The diversity of Kv2 current is increased by interacting with the electrically silent Kv (KvS) subunits Kv5−Kv6 and Kv8−Kv9, into functional heterotetrameric Kv2/KvS channels. These Kv2/KvS channels possess unique biophysical properties and display a more tissue-specific expression pattern, making them more desirable pharmacological and therapeutic targets. However, little is known about the pharmacological properties of these heterotetrameric complexes. We demonstrate that Kv5.1, Kv8.1 and Kv9.3 currents were inhibited differently by the channel blocker 4−aminopyridine (4−AP) compared to Kv2.1 homotetramers. In contrast, Kv6.4 currents were potentiated by 4−AP while displaying moderately increased affinities for the channel pore blockers quinidine and flecainide. We found that the 4−AP induced potentiation of Kv6.4 currents was caused by modulation of the Kv6.4−mediated closed−state inactivation: suppression by 4−AP of the Kv2.1/Kv6.4 closed−state inactivation recovered a population of Kv2.1/Kv6.4 channels that was inactivated at resting conditions, i.e. at a holding potential of −80 mV. This modulation also resulted in a slower initiation and faster recovery from closed−state inactivation. Using chimeric substitutions between Kv6.4 and Kv9.3 subunits, we demonstrated that the lower half of the S6 domain (S6c) plays a crucial role in the 4−AP induced potentiation. These results demonstrate that KvS subunits modify the pharmacological response of Kv2 subunits when assembled in heterotetramers and illustrate the potential of KvS subunits to provide unique pharmacological properties to the heterotetramers, as is the case for 4−AP on Kv2.1/Kv6.4 channels.

K-v channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10-20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker's sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a alpha-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.

Widely acknowledged to be the first person to make detailed studies of microscopic objects, the pioneering work of Antonie van Leeuwenhoek (1632-1723) provided the first glimpse of neurons when he observed sections of bovine optic nerves (van Zuylen, 1981). It was over a century later when the Italian scientist Camillo Golgi (1843-1926) developed la reazione nera (‘the black reaction’), later dubbed the Golgi stain or Golgi method, in 1873. This approach revealed a hitherto unseen world, one brought to light in stunning fashion in the decades that followed by Santiago Ramon y Cajal (1852-1934), who refined Golgi’s techniques and published numerous articles detailing the fine structure of the nervous system (Ramon y Cajal, 1995). The inescapable conclusion of this work that the nervous system is composed of discrete cellular units, or neurons, that communicate with one another in a vast network came to be known as ‘the neuron doctrine.’ It is from these initial findings that the sophisticated techniques available to today’s scientists are descended.

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The kappa-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca2+-activated K+ channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC50 = 9.9 +/- 1.6 mu M). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the kappa-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new kappa-KTx5 subfamily. (C) 2012 Elsevier Inc. All rights reserved.

Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.

International audience; Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1-S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5-S6) is surrounded by four voltage sensor domains (S1-S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5(L)) and S6 C terminus (S6(T)). From these data, we hypothesized that S4S5(L) behaves like a ligand specifically interacting with S6(T) and stabilizing the closed state. To test this hypothesis, we designed plasmid-encoded peptides corresponding to portions of S4S5(L) and S6(T) of the voltage-gated potassium channel KCNQ1 and evaluated their effects on the channel activity in the presence and absence of the ancillary subunit KCNE1. We showed that S4S5(L) peptides inhibit KCNQ1, in a reversible and state-dependent manner. S4S5(L) peptides also inhibited a voltage-independent KCNQ1 mutant. This inhibition was competitively prevented by a peptide mimicking S6(T), consistent with S4S5(L) binding to S6(T). Val(254) in S4S5(L) is known to contact Leu(353) in S6(T) when the channel is closed, and mutations of these residues alter the coupling between the two regions. The same mutations introduced in peptides altered their effects, further confirming S4S5(L) binding to S6(T). Our results suggest a mechanistic model in which S4S5(L) acts as a voltage-dependent ligand bound to its receptor on S6 at rest. This interaction locks the channel in a closed state. Upon plasma membrane depolarization, S4 pulls S4S5(L) away from S6(T), allowing channel opening.

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Due to entangled results concerning K v 1 subunit distribution in the gastrointestinal wall, we aimed to unravel the expression of the delayed rectifier potassium subunits K v 1.1 and K v 1.2 in the murine ileum. Presence and distribution of both subunits were determined in cryosections and whole-mount preparations of the ileum of three different murine strains by indirect immunofluorescence, and analysed by conventional fluorescence and confocal microscopy. Distribution of both subunits was similar in the ileum of the three strains. K v 1.1 immunoreactivity (IR) was found in some S100-expressing enteroglial cells (EGC) located at the periphery of myenteric ganglia, in S100-positive EGC along interganglionic, intramuscular and vascular nerve fibres, and in S100-positive EGC of the submucous plexus. K v 1.1 IR was also observed in some GFAP-expressing EGC at the periphery of myenteric ganglia, and in GFAP-positive EGC of submucous ganglia. K v 1.2 IR was detected in some intramuscular S100-positive EGC, in almost all submucous S100-expressing EGC, and in a few GFAP-expressing EGC. K v 1.2 IR was also expressed in a majority of enteric neurons. Coding of these neurons showed that all cholinergic and most nitrergic neurons express K v 1.2. In conclusion, the results showed that K v 1.1 and K v 1.2 were predominantly expressed in distinct EGC phenotypes. K v 1.2 was also observed in distinct neuron subpopulations. Our results support the active role of EGC with distinct phenotypes in intestinal functions, which is relevant in view of their modulating role on intestinal barrier and inflammatory responses.

Silent voltage-gated K+ (Kv) subunits interact with Kv2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both Kv2 and silent Kv subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of Kv2.1, Kv2.2, and silent subunit Kv6.1, Kv8.1, Kv9.1, Kv9.2, and Kv9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of Kv2.x and Kv9.x subunits in cultured small DRG neurons. To investigate if Kv2 and silent Kv subunits are underlying the delayed rectifier K+ current ( IK) in these neurons, Kv2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of Kv2 antibodies. Both ScTx- and anti-Kv2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of IK. A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric Kv2.1 channels, whereas the other component represents heterotetrameric Kv2.1/silent Kv channels. These observations support a physiological role for silent Kv subunits in small DRG neurons.

Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments.

Noise-induced hearing loss (NIHL) is one of the most important occupational diseases and, after presbyacusis, the most frequent cause of hearing loss. NIHL is a complex disease caused by an interaction between environmental and genetic factors. The various environmental factors involved in NIHL have been relatively extensively studied. On the other hand, little research has been performed on the genetic factors responsible for NIHL. To test whether the variation in genes involved in coupling of cells and potassium recycling in the inner ear might partly explain the variability in susceptibility to noise, we performed a case-control association study using 35 SNPs selected in 10 candidate genes on a total of 218 samples selected from a population of 1,261 Swedish male noise-exposed workers. We have obtained significant differences between susceptible and resistant individuals for the allele, genotype, and haplotype frequencies for three SNPs of the KCNE1 gene, and for the allele frequencies for one SNP of KCNQ1 and one SNP of KCNQ4. Patch-clamp experiments in high K+-concentrations using a Chinese hamster ovary (CHO) cell model were performed to investigate the possibility that the KCNE1-p.85N variant (NT_011512.10:g.21483550G>A; NP_00210.2:p.Asp85Asn) was causative for high noise susceptibility. The normalized current density generated by KCNQ1/KCNE1-p.85N channels, thus containing the susceptibility variant, differed significantly from that from wild-type channels. Furthermore, the midpoint potential of KCNQ1/KCNE1-p.85N channels (i.e., the voltage at which 50% of the channels are open) differed from that of wild-type channels. Further genetic and physiological studies will be necessary to confirm these findings.

Objective: Mutations in the KCNH2 (hERG, human ether-a-go-go related gene) gene may cause a reduction of the delayed rectifier current I-Kr, thereby leading to the long QT syndrome (LQTS). The reduced I-Kr delays the repolarisation of cardiac cells and renders patients vulnerable to ventricular arrhythmias and sudden death. We identified a novel mutation in a LQTS family and investigated its functional consequences using molecular and microscopic techniques. Methods and results: Genetic screening in the LQTS family revealed a heterozygous frameshift mutation p.Pro872fs located in the C-terminus of the KCNH2 gene. The mutation leads to a premature truncation of the C-terminus of the hERG protein. p.Pro872fs channels lack 282 amino acids at the C-terminus and possess an extra 4-amino acid tail. Both the kinetic and biochemical properties of the p.Pro872fs and p.Pro872fs/WT channels were studied in HEK293 cells and resulted in a novel proof of concept for heterozygous LQTS mutations: homotetrameric p.Pro872fs channels displayed near-normal expression, trafficking, and channel kinetics. Unexpectedly, upon co-expression of p.Pro872fs and WT channels, the repolarising power (the proportion of hERG current contributing to the action potential as the percentage of the total current available) was substantially higher during action potential clamp experiments as compared to WT channels alone. This would lead to a shorter rather than a prolonged QT interval. However, at the same time, heterotetramerisation of p.Pro872fs and WT channels also caused a dominant negative effect on trafficking by an increase in ER retention of these heterotetrameric channels, which surpassed the former gain in repolarising power. Conclusion: The LQTS phenotype in the studied family is caused by a mutation with novel properties. We demonstrate that a KCNH2 mutation that clinically leads to long QT syndrome causes at the cellular level both a "gain" and a "loss" of HERG channel function due to a kinetic increase in repolarising power and a decrease in trafficking efficiency of heteromultimeric channels. (C) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved

A novel mutation (T65P) in the PAS domain of the human potassium channel HERG results in the long QT syndrome by trafficking deficiency.Paulussen A, Raes A, Matthijs G, Snyders DJ, Cohen N, Aerssens J.Department of Pharmacogenomics, Johnson & Johnson Pharmaceutical Research and Development, Beerse B-2340, Belgium. paulussen.aimee@advalvas.beThe congenital long QT syndrome is a cardiac disease characterized by an increased susceptibility to ventricular arrhythmias. The clinical hallmark is a prolongation of the QT interval, which reflects a delay in repolarization caused by mutations in cardiac ion channel genes. Mutations in the HERG (human ether-a-go-go-related gene KCNH2 can cause a reduction in I(Kr), one of the currents responsible for cardiac repolarization. We describe the identification and characterization of a novel missense mutation T65P in the PAS (Per-Arnt-Sim) domain of HERG, resulting in defective trafficking of the protein to the cell membrane. Defective folding of the mutant protein could be restored by decreased cell incubation temperature and pharmacologically by cisapride and E-4031. When trafficking was restored by growing cells at 27 degrees C, the kinetics of the mutated channel resembled that of wild-type channels although the rate of activation, deactivation, and recovery from inactivation were accelerated. No positive evidence for the formation of heterotetramers was obtained by co-expression of wild-type with mutant subunits at 37 degrees C. As a consequence the clinical symptoms may be explained rather by haploinsufficiency than by dominant negative effects. This study is the first to relate a PAS domain mutation in HERG to a trafficking deficiency at body temperature, apart from effects on channel deactivation

Voltage-gated K + channels control excitability in neuronal and various other tissues. We identified three unique α-subunits of voltage-gated K + -channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K + channel subunits, they did not produce K + currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with “silent subunits” to diversify its function. Including the subunits described here, the “silent subunits” represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.

[Objective]: The goal of this study was to characterize the electrophysiological properties of the Kv4.3 channels expressed in a mammalian cell line. [Methods]: Currents were recorded using the whole-cell voltage clamp technique. [Results]: The threshold for activation of the expressed Kv4.3 current was approximately −30 mV. The dominant time constant for activation was 1.71±0.16 ms (n=10) at +60 mV. The current inactivated, this process being incomplete, resulting in a sustained level which contributed 15±2% (n=25) of the total current. The time course of inactivation was fit by a biexponential function, the fast component contributing 74±5% (n=9) to the overall inactivation. The fast time constant was voltage-dependent [27.6±2.0 ms at +60 mV (n=10) versus 64.0±3.6 ms at 0 mV (n=10); P0.05]. The voltage-dependence of inactivation exhibited midpoint and slope values of −26.9±1.5 mV and 5.9±0.3 mV (n=21). Recovery from inactivation was faster at more negative membrane potentials [203±17 ms (n=13) and 170±19 ms (n=4), at −90 and −100 mV]. Bupivacaine block of Kv4.3 channels was not stereoselective (KD∼31 μM). [Conclusions]: The functional profile of Kv4.3 channels expressed in Ltk− cells corresponds closely to rat ITO, although differences in recovery do not rule out association with accessory subunits. Nevertheless, the sustained component needs to be considered with respect to native ITO., This work has been supported by HL47599 (D.J.S.), HL49330 (M.M.T.), HL46681 (D.J.S. and M.M.T.) from the National Institutes of Health, and by FIS 95/0318 (C.V.), CICYT SAF96-0042 (J.T.) and CICYT SAF98-0058 (C.V.) Grants.

Objective: The aim of this study was to analyze the effects of a neutral local anaesthetic, benzocaine, on a cardiac K+ channel cloned from human ventricle. Methods: Experiments were performed on hKv1.5 channels stably expressed on mouse cells using the whole-cell configuration of the patch clamp technique. Results: At 10 nM, benzocaine increased the current amplitude (“agonist effect”) by shifting the activation curve 8.4±2.7 mV in the negative direction, and slowed the time course of tail current decline. In contrast, benzocaine (100–700 μM) inhibited hKv1.5 currents ( K D=901±81 μM), modified the voltage-dependence of channel activation, which became biphasic, and accelerated the channel deactivation. Extracellular K+ concentration ([K+]o) also affected the channel gating. At 140 mM [K+]o, the time course of tail currents deactivation was significantly accelerated, whereas at 0 mM [K+]o, it was slowed. At both [K+]o the activation curve became biphasic. Benzocaine accelerated the tail current decay at 0 mM but not at 140 mM [K+]o. The reduction in the permeation of K+ through the pore did not modify the blocking effects of micromolar concentrations of benzocaine, but suppressed the agonist effect observed at nanomolar concentrations. Conclusions: All these results suggest that benzocaine blocks and modifies the voltage- and time-dependent properties of hKv1.5 channels, binding to an extracellular and to an intracellular site at the channel level. Moreover, both sites are related to each other and can also interact with K+.

The Kvbeta1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 +/- 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvbeta1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvbeta1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvbeta1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvbeta1.3-mediated effects involve at least two distinct domains of this beta-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvbeta1.3-induced shift in the activation midpoint is functionally distinct from inactivation.

We have cloned HERG USO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is approximately 2-fold more abundant than that for HERG1 (the originally described cDNA). After transfection of HERG USO in Ltk- cells, no current was observed. However, coexpression of HERG USO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO, HERGDeltaC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGDeltaC, indicating that the C terminus of HERG1 includes a domain (/=104 amino acids) that is critical for faithful recapitulation of IKr. The lack of this C-terminal domain not only explains the finding that HERG USO does not generate IKr but also indicates a similar mechanism for hitherto-uncharacterized long QT syndrome HERG mutations that disrupt the splice site or the C-terminal. We suggest that the amplitude and gating of cardiac IKr depends on expression of both HERG1 and HERG USO.

Objective: The aim of this study was to analyze the effects of loratadine on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line. Methods: Currents were studied using the whole-cell configuration of the patch−clamp technique in Ltk− cells transfected with the gene encoding hKv1.5 channels. Results: Loratadine inhibited in a concentration-dependent manner the hKv1.5 current, the apparent affinity being 1.2±0.2 μM. The blockade increased steeply between −30 and 0 mV which corresponded with the voltage range for channel opening, thus suggesting that the drug binds preferentially to the open state of the channel. The apparent association and dissociation rate constants were (3.6±0.5)×106·M−1·s−1 and 3.7±1.6·s−1, respectively. Loratadine, 1 μM, increased the time constant of deactivation of tail currents elicited on return to −40 mV after 500 ms depolarizing pulses to +60 mV from 36.2±3.4 to 64.9±3.6 ms ( n = 6, P

Objective: The transient outward current ( I TO ) plays an important role in early repolarization and overall time course of the cardiac action potential. At least two K+ channel α-subunits cloned from cardiac tissue (Kv1.4 and Kv4.2) encode rapidly inactivating channels. The goal of this study was to determine functional and pharmacological properties of Kv4.2 expressed in mammalian cells, especially those that would differentiate between both isoforms in comparison to native I TO . Methods: Both Kv4.2 and Kv1.4 isoforms were stably expressed in mouse L-cell lines, and expressed currents were studied using whole-cell voltage clamp techniques. Results: The expressed Kv4.2 currents displayed fast inactivation with a half-inactivation potential of −41 mV. Recovery from inactivation was rapid ( τ recov = 160 ms at −90 mV) and strongly voltage-dependent. Flecainide (10 μM) had minimal effects on Kv1.4 currents, but reduced Kv4.2 peak current by 53% and increased the apparent rate of inactivation consistent with open channel block. Quinidine (10–20 μM) reduced the peak current and accelerated the apparent rate of inactivation in both isoforms. The Kv4.2 current displayed use-dependent unblock in the presence of 4-AP. Conclusions: The functional properties of Kv4.2, especially the flecainide sensitivity, resemble those of I TO in rat (and human) myocytes better than those of Kv1.4. These results provide the necessary functional support for the hypothesis that Kv4.2 is a major isoform contributing to cardiac I TO , consistent with independent biochemical and molecular evidence that indicates that Kv4.2 is readily detected in rat myocytes.

The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon. These data indicate 1) the charged form of both bupivacaine enantiomers block the hKv1.5 channel after it opens, 2) binding occurs within the transmembrane electrical field, 3) unbinding is required before the channel can close, 4) block of hKv1.5 channels by bupivacaine is markedly stereoselective, with the R(+)-enantiomer being the more potent one, 5) this stereoselective block was associated with a 1.11 -kcal/mol difference in binding energy between both enantiomers, and 6) the stereoselectivity derives mainly from a difference in the association rate constants, suggesting that the S(-)-enantiomer is less likely to access the binding site in an optimal configuration.

To gain insight into the molecular basis of cardiac repolarization, we have expressed K+ channels cloned from ventricular myocardium in Xenopus oocytes. A recently identified human cardiac K+ channel isoform (human Kv1.4) has properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current. However, these channels recovered from inactivation much slower than native channels. Hybrid channels consisting of subunits from different K+ channel clones (delayed rectifier channels [Kv1.1, Kv1.2, and Kv1.5] and Kv1.4) were created by coinjection of cRNAs in oocytes. Multimeric channels consisting of Kv1.4:Kv1.1, Kv1.4:Kv1.2, and Kv1.4:Kv1.5 were expressed and compared. The hybrid channels displayed characteristics of heterotetrameric channels with kinetics that more closely resembled a native cardiac transient outward current. The inactivation and recovery from inactivation of the heteromeric channels indicated that the presence of a single inactivating subunit (Kv1.4) was probably sufficient to cause channel inactivation. The results demonstrate that expression of different K+ channel genes can produce channel protein subunits that assemble as heteromultimers with unique properties. It is shown that certain combinations of voltage-gated K+ channels probably do not contribute to native transient outward current. However, one combination of subunits could not be excluded. Therefore, this mechanism of channel assembly may underlie some of the functional diversity of potassium channels found in the cardiovascular system.

The C-terminal regions of Kv1 K+ channels show little conservation between isoforms except for the last four C-terminal residues, (E/L)TDV, which are well conserved from Drosophila to man. Deletions of the 4,16, and 57 C-terminal residues of the human Kv1.5 channel did not affect whole cell current amplitude, midpoint of activation, degree of inactivation, or activation kinetics following expression in mouse L-cells. Similar results were obtained with the rat Kv1.1 channel. Therefore, the conserved (E/L)TDV motif, and most of the C-terminal amino acids, are not required for Kv1 channel expression.

Voltage-gated potassium (Kv) channels are tetramers of α-subunits, each composed out of six membrane-spanning helices (S1-S6) with a pore loop between S5 and S6 that forms the channel's selectivity filter. The activation gate that seals off the ion conducting pore in a closed channel is controlled by the transmembrane potential. After channel activation most Kv channels display C-type inactivation, a process that is believed to involve reorientations of the selectivity filter and results in a non-conducting channel although the channel gate is open. hKv1.5 (a Shaker-type Kv channel) displays such C-type inactivation. Here we report that an alanine substitution for residue T480 that is located at the end of the pore-helix prevents hKv1.5 channels from entering the inactivated state. The mutant T480A had an isochronal activation curve similar to Wild Type hKv1.5 when determined with 250ms depolarizing steps. Longer depolarizations (∼5 seconds) caused WT channels to inactivate (∼58%) displaying an inactivation curve with a midpoint of −23.2 ± 1.2 mV and a slope factor of about 4. However, T480A did not inactivate and such long depolarizations caused an additional (slow) activation at more negative potentials thus generating an isochronal activation curve with properties that were similar to the inactivation process in WT channels. To investigate the structural changes that underlie the unusual behaviour of this mutant, we performed a series of MD simulations of the pore domain of WT hKv1.5 and T480A. Analysis of the trajectories shows that T480A affects the stability and flexibility of the filter region and the surrounding pore loop. These results show that residue T480 (located outside the pore region that determines the integrity of the selectivity filter) affects the stability of the filter and influences C-type inactivation.

Voltage-gated K+ (Kv) channels are formed as tetramers of α-subunits. Based on sequence similarity, closely Shaker-related Kv subunits have been divided into eight subfamilies (Kv1-Kv6 and Kv8-Kv9). Members of the Kv1-Kv4 subfamilies form - within the subfamily - functional channels in both homo- and heterotetrameric configuration. The Kv5, Kv6, Kv8 and Kv9 subunits are designated silent Kv (KvS) subunits since they do not form functional homotetrameric channels, but they co-assemble with Kv2 subunits generating functional heterotetrameric Kv2/KvS channel complexes. Kv1-Kv4 channel subunits are thought to assemble as a dimer of dimers, but Kv2.1/Kv9.3 channels have been shown to assemble with a 3:1 stoichiometry - i.e. three Kv2.1 subunits with one Kv9.3 subunit. However, it remains unclear whether this 3:1 stoichiometry can be extrapolated to all Kv2/KvS channels. Here we report that the silent subunit Kv6.4 can assemble with Kv2.1 subunits into heterotetrameric Kv2.1/Kv6.4 channels with a dimer of dimers configuration. Therefore we constructed both Kv2.1-Kv6.4 and Kv6.4-Kv2.1 dimers. Each dimer produced functional channels characterized by a voltage-dependence of inactivation (V1/2 = −59.2 mV and V1/2 = −62.9 mV, respectively) that is shifted approximately 40 mV into hyperpolarized direction compared to the voltage-dependence of inactivation of the Kv2.1-Kv2.1 dimer (V1/2 = −23.5 mV). These voltage-dependencies are similar to those obtained after expression of Kv2.1 monomers alone (V1/2 = −18.2 mV) and after co-expression with Kv6.4 (V1/2 = −55.6 mV). In addition, this dimer of dimers configuration was confirmed with Fluorescence Resonance Energy Transfer (FRET) experiments using YFP and CFP tagged dimers. While these data may not rule out an alternate 3:1 stoichiometry, they do indicate that Kv2.1 and Kv6.4 subunits can heterotetramerize into functional channels with a 2:2 stoichiometry.

Fully assembled voltage-gated potassium (Kv) channel are tetramers of α-subunits. The silent (KvS) channel subunits (Kv5-9) do not form functional homotetramers due to retention in the endoplasmic reticulum (ER). This ER retention is relieved by assembly with Kv2 subunits generating functional Kv2/KvS heterotetramers. For the Kv1-4 subfamilies, the N-terminal T1 domain determines the subfamily specific tetramerization by preventing interactions between subunits that belong to different subfamilies. However, yeast-two-hybrid experiments showed an interaction between the N-termini of Kv6.4 and both Kv2.1 and Kv3.1. We demonstrate here that the subfamily specific Kv2.1/Kv6.4 heterotetramerization is determined by additional C-terminal interactions. Forster Resonance Energy Transfer (FRET) and co-immunoprecipitation (co-IP) using N- and C-terminal Kv6.4 and Kv3.1 fragments as well as N- and C-terminal truncated Kv6.4 and Kv3.1 subunits indicated that both the Kv6.4 N-terminus and S1-S6 segments interact with the corresponding Kv3.1 segments. However, these interactions did not lead to functional Kv3.1/Kv6.4 heterotetramers since co-expression of Kv6.4 with Kv3.1 did not affect the Kv6.4 localization nor the biophysical Kv3.1 properties, and no interaction between Kv3.1 and Kv6.4 could be detected with co-IP experiments using the full length α-subunits. Furthermore, co-expression of Kv3.1 with the (NKv3.1)Kv6.4 chimera - in which the Kv3.1 N-terminus has been introduced in a Kv6.4 background - did also not lead to functional heterotetramers. These results together suggest that the inability of the Kv6.4 C-terminus to associate with its interaction partner inhibits the formation of functional Kv3.1/Kv6.4 heterotetramers. Indeed, FRET and co-IP experiments using N- and C-terminal fragments demonstrated that the C-terminus of Kv6.4 physically interacts with the N-terminus of Kv2.1 but not with the Kv3.1 N-terminus. This indicates that the specific Kv2.1/Kv6.4 heterotetramerization is determined by specific interactions between Kv2.1 and Kv6.4 that involve both the N- and C-termini.

KCNQ1 α-subunits are composed out of six transmembrane segments (S1-S6) that tetramerize into a functional channel. In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac IKs and consequently mutations in KCNQ1 are linked to the congenital LQT1 syndrome. Similar to other Kv channels, the S1-S4 segments form the voltage sensing domain that senses the membrane potential and that controls the opening or closure of the channel gate through an electromechanical coupling. In other channels a direct interaction between the S4-S5 linker and bottom part of S6 has been shown to constitute this electromechanical coupling. We previously identified residues in the C-terminal part of S6 that are critical for KCNQ1 gating. To investigate if these residues interact with the S4-S5 linker, we performed an alanine/tryptophan substitution scan of the S4-S5 linker sequence. Based on their impact on channel gating, we categorized these substitutions as either “high” or “low impact”. The pattern of “high impact” positions was consistent with an α-helical configuration and clustered on one side of the S4-S5 linker. Since substitutions at these positions markedly impaired channel gating, they are good candidates to contact residues in the bottom part of S6. Indeed, replacing valine 254 in the S4-S5 linker by a leucine resulted in channels that were partially constitutively open but channel closure could be rescued by combining V254L with the S6 mutation L353A that by itself displayed a similar phenotype as V254L. The observation that all known LQT1 mutations in the S4-S5 linker map on the “high impact” side further strengthens the proposal that this face of the S4-S5 linker contacts the C-terminal S6 segment and constitutes part of the electromechanical coupling in KCNQ1 channels.

Voltage-gated potassium (Kv) channels are tetramers of 6-transmembrane domain (S1-S6) α-subunits. The activation gate that seals off the ion conducting pore is under control of the voltage-sensing domain and locates at the bundle crossing of the S6 segments. After channel opening most Kv channels display slow inactivation, a process that involves rearrangements of the selectivity filter (SF) resulting in a non-conducting channel although the S6-gate is open. Recent evidence argued for a strong coupling between inactivation and activation: after gate opening the S6 segment undergoes structural rearrangements that would be transmitted up to the level of the SF destabilizing its conformation. Substituting in Kv1.5 residue T480 that locates at the bottom of the SF by an alanine generated mutant channels which instead of inactivating displayed a second open state, characterised by slowly increasing current. Several molecular dynamics (MD) simulations showed a MD trajectory that displays a hydrophobic collapse of the central cavity with S6 dynamics decreasing the pore radius of the S6-gate. The simulations further showed that the SF did constrict in WT channels but not in the mutant. Ionic current measurements of the mutant T480A channels showed that after prolonged depolarizations - pushing the channels into the second conducting state - the activation gate could close and reopen with the channel remaining in the second conducting state. To convert the channels back to their original conducting state longer repolarizing times were needed. Thus, using the T480A pore mutant we could directly determine from ionic currents that gate closure in Kv1.5 channels does not depend on the status of the SF which, as suggested by Deutsch et al. implies the existence of a channel state with both a closed gate and inactivated selectivity filter. (Support: FWO-G025608)