Ks Channel Research Articles

Abstract Mo063: Hacking the ubiquitin code to distinctively modulate ion channel functional expression

Ubiquitin is a powerful modulator of ion channel fate, yet the mechanistic basis and scope of its regulation has remained elusive due to the complexity of the ubiquitin code and an inability to achieve substrate-specific control of the ubiquitome. To circumvent this and to specifically interrogate the role of distinct ubiquitin linkages on the cardiac repolarizing I Ks channel KCNQ1 (Q1), we developed a toolkit of engineered deubiquitinases (enDUBs) featuring a GFP/YFP-targeted nanobody fused to catalytic domains of DUBs with preferences for hydrolyzing distinctive polyubiquitin linkage types: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K27/K33; and USP21 - non-specific. Co-expressing each of the enDUBs with YFP-tagged Q1 channels in HEK293 cells led to decreased channel ubiquitination but produced quantitative and qualitative differences in Q1 expression. NanoCezanne and nanoTRABID significantly increased Q1 surface density and ionic currents, whereas nanoOTUD1 and nanoUSP21 yielded more moderate effects. In sharp contrast, nanoOTUD4 downregulated Q1 surface density and currents. The impact on steady-state Q1 surface density was achieved by divergent trafficking mechanisms – nanoTRABID and nanoCezanne increased channel forward trafficking to the surface, while nanoOTUD4 reduced channel forward trafficking. The E3 ligases NEDD4L and ITCH both eliminate Q1 functional expression but with different polyubiquitin signatures as assessed by mass spectrometry. The enDUBs nanoOTUD1, nanoCezanne and nanoUSP21 protected Q1 from NEDD4L; whereas nanoTRABID, nanoOTUD1, nanoCezanne and nanoUSP21 shielded Q1 from ITCH. In adult guinea ventricular cardiomyocytes, the enDUBs displayed context-specific regulatory effects, where nanoOTUD1, nanoCezanne and nanoUSP21 significantly enhanced Q1 surface expression and stability, with nanoTRABID having more moderate effects, while nanoOTUD4 sharply decreased Q1 trafficking to the sarcolemma. Finally, linkage-specific enDUBs demonstrated varying capabilities to rescue distinct trafficking-deficient mutant Q1 channels that cause long QT syndrome and predispose to sudden cardiac death. The results reveal a rich multi-faceted role for linkage-specific ubiquitination in regulating ion channel trafficking with implications to develop targeted therapies for trafficking-deficit ion channelopathies.

Dopamine-Mediated Major Depressive Disorder in the Neural Circuit of Ventral Tegmental Area-Nucleus Accumbens-Medial Prefrontal Cortex: From Biological Evidence to Computational Models.

Major depressive disorder (MDD) is a serious psychiatric disorder, with an increasing incidence in recent years. The abnormal dopaminergic pathways of the midbrain cortical and limbic system are the key pathological regions of MDD, particularly the ventral tegmental area- nucleus accumbens- medial prefrontal cortex (VTA-NAc-mPFC) neural circuit. MDD usually occurs with the dysfunction of dopaminergic neurons in VTA, which decreases the dopamine concentration and metabolic rate in NAc/mPFC brain regions. However, it has not been fully explained how abnormal dopamine concentration levels affect this neural circuit dynamically through the modulations of ion channels and synaptic activities. We used Hodgkin-Huxley and dynamical receptor binding model to establish this network, which can quantitatively explain neural activity patterns observed in MDD with different dopamine concentrations by changing the kinetics of some ion channels. The simulation replicated some important pathological patterns of MDD at the level of neurons and circuits with low dopamine concentration, such as the decreased action potential frequency in pyramidal neurons of mPFC with significantly reduced burst firing frequency. The calculation results also revealed that NaP and KS channels of mPFC pyramidal neurons played key roles in the functional regulation of this neural circuit. In addition, we analyzed the synaptic currents and local field potentials to explain the mechanism of MDD from the perspective of dysfunction of excitation-inhibition balance, especially the disinhibition effect in the network. The significance of this article is that we built the first computational model to illuminate the effect of dopamine concentrations for the NAc-mPFC-VTA circuit between MDD and normal groups, which can be used to quantitatively explain the results of existing physiological experiments, predict the results for unperformed experiments and screen possible drug targets.

Modulation of IKs channel-PIP2 interaction by PRMT1 plays a critical role in the control of cardiac repolarization.

Recent studies have shown that protein arginine methyltransferase 1 (PRMT1) is highly expressed in the human heart, and loss of PRMT1 contributes to cardiac remodeling in the heart failure. However, the functional importance of PRMT1 in cardiac ion channels remains uncertain. The slow activating delayed rectifier K+ (I Ks) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits and is a new therapeutic target for treating lethal arrhythmias in many cardiac pathologies, especially heart failure. Here, we demonstrate that PRMT1 is a critical regulator of the I Ks channel and cardiac rhythm. In the guinea pig ventricular myocytes, treatment with furamidine, a PRMT1‐specific inhibitor, prolonged the action potential duration (APD). We further show that this APD prolongation was attributable to I Ks reduction. In HEK293T cells expressing human KCNQ1 and KCNE1, inhibiting PRMT1 via furamidine reduced I Ks and concurrently decreased the arginine methylation of KCNQ1, a pore‐forming α‐subunit. Evidence presented here indicates that furamidine decreased I Ks mainly by lowering the affinity of I Ks channels for the membrane phospholipid, phosphatidylinositol 4,5‐bisphosphate (PIP2), which is crucial for pore opening. Finally, applying exogenous PIP2 to cardiomyocytes prevented the furamidine‐induced I Ks reduction and APD prolongation. Taken together, these results indicate that PRMT1 positively regulated I Ks activity through channel–PIP2 interaction, thereby restricting excessive cardiac action potential.

Effects of allocryptopine on outward potassium current and slow delayed rectifier potassium current in rabbit myocardium.

ObjectiveAllocryptopine (ALL) is an effective alkaloid of Corydalis decumbens (Thunb.) Pers. Papaveraceae and has proved to be anti-arrhythmic. The purpose of our study is to investigate the effects of ALL on transmural repolarizing ionic ingredients of outward potassium current (Ito) and slow delayed rectifier potassium current (IKs).MethodsThe monophasic action potential (MAP) technique was used to record the MAP duration of the epicardium (Epi), myocardium (M) and endocardium (Endo) of the rabbit heart and the whole cell patch clamp was used to record Ito and IKs in cardiomyocytes of Epi, M and Endo layers that were isolated from rabbit ventricles.ResultsThe effects of ALL on MAP of Epi, M and Endo layers were disequilibrium. ALL could effectively reduce the transmural dispersion of repolarization (TDR) in rabbit transmural ventricular wall. ALL decreased the current densities of Ito and IKs in a voltage and concentration dependent way and narrowed the repolarizing differences among three layers. The analysis of gating kinetics showed ALL accelerated the channel activation of Ito in M layers and partly inhibit the channel openings of Ito in Epi, M and Endo cells. On the other hand, ALL mainly slowed channel deactivation of IKs channel in Epi and Endo layers without affecting its activation.ConclusionsOur study gives partially explanation about the mechanisms of transmural inhibition of Ito and IKs channels by ALL in rabbit myocardium. These findings provide novel perspective regarding the anti-arrhythmogenesis application of ALL in clinical settings.

Computational simulations of the effects of the G229D KCNQ1 mutation on human atrial fibrillation.

Atrial fibrillation (AF) is related to mutations at the genetic level. This includes mutations in genes that encode KCNQ1, a subunit of the I Ks channel. Here, we investigate the mechanism of gain-of-function in I Ks towards the occurrence of AF. We used the Courtemanche-Ramirez-Nattel (CRN) human atrial cell model (Am J Physiol Heart Circ Physiol 275:H301-H321, 1998) and applied the modification proposed by Hasegawa et al. (Heart Rhythm 11:67-75, 2014) to fit the behavior of I Ks due to the G229D mutation in KCNQ1 under a heterozygous mutant form. This was incorporated into two-(2D) and three-dimensional (3D) tissue models, where the mutation sustained a reentrant wave. However, under the wild-type condition, the reentrant wave terminated before the end of our simulations (in 2D, the spiral wave terminated before 10s, while in 3D, the spiral wave terminated before 13s). Sustained reentry under the mutation conditions also resulted in a spiral wave breakup in the 3D model, which was sustained until the end of the simulation (20s), indicating AF.

N-Arachidonoyl Taurine Rescues Diverse Long QT Syndrome-Associated Mutations in the Cardiac I←Ks Channel

The cardiac I←Ks channel is important for cardiomyocyte repolarization. More than 300 loss-of-function mutations in the genes encoding I←Ks have been identified in patients with Long QT syndrome. These mutations cause cardiac arrhythmias, such as torsades de pointes and ventricular fibrillation. How specific mutations cause arrhythmia is, however, not known in most cases and there is no approved I←Ks channel activator for treatment of arrhythmia. In this work, we study the biophysical properties and mechanism of loss of function of Long QT syndrome-associated I←Ks channel mutations expressed in Xenopus laevis oocytes. We next test the ability of the fatty acid analogue N-arachidonoyl taurine to restore the function of these mutants. Using two-electrode voltage clamp, we find that a number of Long QT syndrome-associated, loss-of-function I←Ks channel mutations shift the voltage dependence of opening towards positive voltages and speed up channel closing. Using voltage clamp fluorometry, we show that these alterations are caused by either disrupted voltage sensor movement or gate opening. We also find that the fatty acid analogue N-arachidonoyl taurine activates all tested Long QT syndrome-associated I←Ks channel mutations by shifting their voltage dependence of activation towards more negative voltages and altering the kinetics of channel opening and closing. Thus, N-arachidonoyl taurine restores the function of these mutants and compensates for the loss of function induced by these mutations, whether the mutations disrupted voltage sensor movement or gate opening. N-AT also shortens a drug-induced prolonged QT interval in isolated perfused guinea pig hearts in Langendorff preparation back to a normal range. N-AT is therefore a promising future I←Ks channel activator that may restore a physiological QT interval by enhancing the function of diverse Long QT syndrome I←Ks mutants, independent of the mutational defect.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism

Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

A KCNE1 C-Terminus Long QT Mutation Disrupts a Crucial Interaction with the Kv7.1 Coiled-Coil Helix C and Reduces I KS Channel Expression

Kv7.1 α-subunit assembles with the KCNE1 auxiliary subunit to form the cardiac IKS K+ channel. Mutations in these subunit genes produce the long QT syndrome, a life-threatening ventricular arrhythmia. We recently explored the direct interactions between the C-termini of Kv7.1 and KCNE1 using purified recombinant proteins in a series of in-vitro pull-down experiments. We found that the KCNE1 C-terminus physically interacts with the coiled-coil helix-C of the tetramerization domain. Here we show that the missense LQT5 mutation (P127T) located in the KCNE1 distal C-terminus and previously found to produce a dominant Romano-Ward LQT syndrome, disrupted the physical interaction of KCNE1 with the Kv7.1 coiled-coil helix C. When co-expressed in CHO cells with WT Kv7.1, the KCNE1 LQT mutant P127T dramatically reduced IKS current density, without altering channel gating properties. Our results show that deletion of the proximal KCNE1 C-terminus (KCNE1Δ69-77) significantly enhanced the binding of KCNE1 to Kv7.1 C-terminus. In contrast, deletions of the more distal regions of KCNE1 C-terminus (KCNE1Δ78-129 and KCNE1Δ109-129) totally prevented its binding to the Kv7.1 C-terminal region. Our data suggest that the most distal region of the KCNE1 C-terminus (aa.109-129) is crucial for the KCNE1 interaction with the Kv7.1 coiled-coil helix-C and that the missense LQT5 mutation P127T located within this domain affects IKS channel expression by disrupting this coiled-coil interaction and possibly disturbing channel trafficking.

Blockade of the I Ks potassium channel: An overlooked cardiovascular liability in drug safety screening?

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I Ks channel, which along with the transient outward current (I to) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I Ks blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I Ks channel blockade and describe the discovery of a potent I Ks blocker whose activity was not detected by standard hERG or in vitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) in vivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I Ks blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I Ks blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity. Assay Drug Development Technology 5, 617–627]). Because of the presence of I Ks channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I Ks blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I Ks liability before testing in humans. The possibility of undetected I Ks blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427–1438] in the ICH S7B regulatory guidelines.

Mechanisms of β-Adrenergic Modulation of IKs in the Guinea-Pig Ventricle: Insights from Experimental and Model-Based Analysis

Detailed understanding of I Ks gating complexity may provide clues regarding the mechanisms of repolarization instability and the resulting arrhythmias. We developed and tested a kinetic model to interpret physiologically relevant I Ks properties, including pause-dependence and modulation by β-adrenergic receptors ( β-AR). I Ks gating was evaluated in guinea-pig ventricular myocytes at 36°C in control and during β-AR stimulation (0.1 μmol/L isoprenaline (ISO)). We tested voltage dependence of steady-state conductance (Gss), voltage dependence of activation and deactivation time constants ( τ act, τ deact), and pause-dependence of τ act during repetitive activations ( τ react). The I Ks model was developed from the Silva and Rudy formulation. Parameters were optimized on control and ISO experimental data, respectively. ISO strongly increased Gss and its voltage dependence, changed the voltage dependence of τ act and τ deact, and modified the pause-dependence of τ react. A single set of model parameters reproduced all experimental data in control. Modification of only three transition rates led to a second set of parameters suitable to fit all ISO data. Channel unitary conductance and density were unchanged in the model, thus implying increased open probability as the mechanism of ISO-induced Gss enhancement. The new I Ks model was applied to analyze ISO effect on repolarization rate-dependence. I Ks kinetics and its β-AR modulation were entirely reproduced by a single Markov chain of transitions (for each channel monomer). Model-based analysis suggests that complete opening of I Ks channels within a physiological range of potentials requires concomitant β-AR stimulation. Transient redistribution of state occupancy, in addition to direct modulation of transition rates, may underlie β-AR modulation of I Ks time dependence.

Tanshinone IIA: A new activator of human cardiac KCNQ1/KCNE1 ( IKs) potassium channels

Tanshinone IIA, one of the main active components from Chinese herb Danshen, is widely used to treat cardiovascular diseases including arrhythmia in Asian countries especially in China. However, the mechanisms underlying its anti-arrythmia effects are not clear. In this study we investigate the effects of tanshinone IIA on human KCNQ1/KCNE1 potassium channels ( I Ks), human ether-a-go-go-related gene potassium channels (hERG), Kv1.5 potassium channels, inward rectifier potassium channels ( I K1) expressed in HEK 293 cells using patch clamp technique. Tanshinone IIA potently and reversibly enhanced the amplitude of I Ks in a concentration dependent manner with an EC 50 of 64.5 μM, accelerated the activation rate of I Ks channels, decelerated their deactivation and shifted the voltage dependence of I Ks activation to negative direction. Isoproteronol, a stimulator of β-adrenergic receptor, at 1 μM and sodium nitroprusside (SNP), a NO donor, at 1 mM, had no significant effects on the enhancement of I Ks by 30 μM tanshinone IIA. N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a selective protein kinase A inhibitor, at 0.1 μM and 1 H-(1,2,4)oxadiazolo(4,3- a)quinoxalin-1-one (ODQ), a selective nitric oxide-sensitive guanylyl cyclase inhibitor, at 10 μM, also had no significant effects on the enhancement of I Ks by 30 μM tanshinone IIA. Tanshinone IIA did not affect expressed hERG channels, Kv1.5 channels and I K1 channels. These results indicate that tanshinone IIA directly and specifically activate human cardiac KCNQ1/KCNE1 potassium channels ( I Ks) in HEK 293 cell through affecting the channels' kinetics.

Contribution of L-Type Ca2+ Channels to Early Afterdepolarizations Induced by I Kr and I Ks Channel Suppression in Guinea Pig Ventricular Myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse

Inherited gain-of-function mutations of genes coding for subunits of the heart slow potassium (I Ks) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that beta-adrenergic receptor (beta-AR)-mediated regulation of the I Ks channel, a natural gain-of-function pathway, can also lead to AF. Using a transgenic I Ks channel mouse model, we studied the role of the channel and its regulation by beta-AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes I Ks channel transgenic mice but not wild-type (WT) littermates that lack I Ks to prolonged atrial arrhythmias. Patch-clamp analysis demonstrated expression and isoprenaline-mediated regulation of I Ks in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that beta-AR stimulation-dependent accumulation of open I Ks channels accounts for the pro-arrhythmic substrate. Our results provide evidence that beta-AR-regulated I Ks channels can play a role in AF and imply that specific I Ks deregulation, perhaps through disruption of the I Ks macromolecular complex necessary for beta-AR-mediated I Ks channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia.

Involvement of tyrosine kinase in the hyposmotic stimulation of I Ks in guinea-pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the hyposmotic stimulation of cardiac I Ks, a slowly activating delayed-rectifier K+ current that promotes repolarization of the action potential. The current was recorded from whole-cell-configured guinea-pig ventricular myocytes before, during, and after their exposure to solution whose osmolarity was 0.75 times normal. Exposure to hyposmotic solution caused a near-doubling of the amplitude of I Ks, with little change in the voltage dependence of current activation. Stable, hyposmotically stimulated I Ks (I Ks,Hypo) was decreased by broadspectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC50 approximately 5 microM) and tyrphostin A25 (IC50 15.8 +/- 1.6 microM) but not by TK-inactive tyrphostin analogs, suggesting that tyrosine phosphorylation is important for maintenance of the current. In agreement with that view, we found that the TK-inhibitor action on I Ks,Hypo was strongly antagonized by vanadate compounds known to inhibit phosphotyrosyl phosphatase. When myocytes were pretreated with TK inhibitors, the stimulation of I Ks was attenuated in a concentration-dependent manner. The attenuation was not due to concomitant attenuation of a stimulation of tyrosine phosphorylation because neither the stimulation of I Ks nor its rate of decay following removal of hyposmotic solution was affected by pretreatment with vanadates. We suggest that the stimulation of I Ks by hyposmotic solution is dependent on a basal tyrosine phosphorylation that modulates a swelling-induced I Ks-stimulatory signal and/or the receptivity of Ks channels to that signal.

Impact of ancillary subunits on ventricular repolarization

Voltage-gated potassium (Kv) channels generate the outward K + ion currents that constitute the primary force in ventricular repolarization. Voltage-gated potassium channels comprise tetramers of pore-forming α subunits and, in probably most cases in vivo, ancillary or β subunits that help define the properties of the Kv current generated. Ancillary subunits can be broadly categorized as cytoplasmic or transmembrane and can modify Kv channel trafficking, conductance, gating, ion selectivity, regulation, and pharmacology. Because of their often profound effects on Kv channel function, studies of the molecular correlates of ventricular repolarization must take into account ancillary subunits as well as α subunits. Cytoplasmic ancillary subunits include the Kv β subunits, which regulate a range of Kv channels and may link channel gating to redox potential, and the KChIPs, which appear most often associated with Kv4 subfamily channels that generate the ventricular I to current. Transmembrane ancillary subunits include the MinK-related proteins (MiRPs) encoded by KCNE genes, which modulate members of most Kv α subunit subfamilies, and the putative 12-transmembrane domain KCR1 protein, which modulates hERG. In some cases, such as the ventricular I Ks channel complex, it is well established that the KCNQ1 α subunit must coassemble with the MinK (KCNE1) single-transmembrane domain ancillary subunit for recapitulation of the characteristic, unusually slowly-activating I Ks current. In other cases, it is not so clear-cut, and in particular, the roles of the other MiRPs (1-4) in regulating cardiac Kv channels such as KCNQ1 and hERG in vivo are under debate. MiRP1 alters hERG function and pharmacology, and inherited MiRP1 mutations are associated with inherited and acquired arrhythmias, but controversy exists over the native role of MiRP1 in regulating hERG (and therefore ventricular I Kr) in vivo. Some ancillary subunits may exhibit varied expression to shape spatial Kv current variation, for example, KChIP2 and the epicardial-endocardial I to current density gradient. Indeed, it is likely that most native ventricular Kv channels exhibit temporal and spatial heterogeneity of subunit composition, complicating both modeling of their functional impact on the ventricular action potential and design of specific current-targeted compounds. Here, we discuss current thinking and lines of experimentation aimed at resolving the complexities of the Kv channel complexes that repolarize the human ventricular myocardium.

Serial Perturbation of MinK in I Ks Implies an α-Helical Transmembrane Span Traversing the Channel Corpus

I Ks channels contain four pore-forming KCNQ1 subunits and two accessory MinK subunits. MinK influences surface expression, voltage-dependence of gating, conduction, and pharmacology to yield the attributes characteristic of native channels in heart. The structure and location of the MinK transmembrane domain (TMD) remains a matter of scrutiny. As perturbation of gating analysis has correctly inferred the peripheral location and α-helical nature of TMDs in pore-forming subunits, the method is applied here to human MinK. Tryptophan and Asparagine substitution at 23 consecutive sites yields perturbation with α-helical periodicity (residues 44–56) followed by an alternating impact pattern (residues 56–63). Arginine substitution across the span suggests that as few as eight sites are occluded from aqueous solution (residues 50–57). We favor a TMD model that is α-helical with the external portion of the span at a lipid-protein boundary and the inner portion within the channel corpus in complex interactions.

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac I Ks

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K + current (I Ks) via unknown mechanisms. In the present study, I Ks was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10–100 μM H89; 200 μM H8; 50 μM H7; 1 μM bisindolylmaleimide I; 10 μM LY294002; 50 μM PD98059), (ii) stimulate serine/threonine kinase activity (1–5 μM forskolin; 0.1 μM phorbol-12-myristate-13-acetate; 10 μM acetylcholine; 0.1 μM angiotensin II; 20 μM ATP), (iii) suppress G-protein activation (10 mM GDPβS), or (iv) disrupt the cytoskeleton (10 μM cytochalasin D), had little effect on the stimulation of I Ks by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 μM) strongly attenuated both the hyposmotic stimulation of I Ks in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.

Tyrosine kinase and phosphatase regulation of slow delayed‐rectifier K+ current in guinea‐pig ventricular myocytes

The objective of this study was to investigate the involvement of tyrosine phosphorylation in the regulation of the cardiac slowly activating delayed-rectifier K(+) current (I(Ks)) that is important for action potential repolarization. Constitutive I(Ks) recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23 (IC(50), 4.1+/-0.6 microm), tyrphostin A25 (IC(50), 12.1+/-2.1 microm) and genistein (IC(50), 64+/-4 microm), but was relatively insensitive to the inactive analogues tyrphostin A1, tyrphostin A63, daidzein and genistin. I(Ks) was unaffected by AG1478 (10 microm), an inhibitor of epidermal growth factor receptor TK, and was strongly suppressed by the Src TK inhibitor PP2 (10 microm) but not by the inactive analogue PP3 (10 microm). The results of experiments with forskolin, H89 and bisindolylmaleimide I indicate that the suppression of I(Ks) by TK inhibitors was not mediated via inhibition of (I(Ks)-stimulatory) protein kinases A and C. To evaluate whether the suppression was related to lowered tyrosine phosphorylation, myocytes were pretreated with TK inhibitors and then exposed to the phosphotyrosyl phosphatase inhibitor orthovanadate (1 mm). Orthovanadate almost completely reversed the suppression of I(Ks) induced by broad-spectrum TK inhibitors at concentrations around their IC(50) values. We conclude that basal I(Ks) is strongly dependent on tyrosine phosphorylation of Ks channel (or channel-regulatory) protein.

Characterization of an LQT5-related mutation in KCNE1, Y81C: Implications for a role of KCNE1 cytoplasmic domain in I Ks channel function

Y81C is a new long QT-5 (LQT5)-related KCNE1 mutation, which is located in the post-transmembrane domain (post-TMD) region in close proximity to three other LQT5 mutations (S74L, D76N, and W87R). We examine the effects of Y81C on the function and drug sensitivity of the slow delayed rectifier channel (I(Ks)) formed by KCNE1 with pore-forming KCNQ1 subunits. We also infer a structural basis for the detrimental effects of Y81C on I(Ks) function. Wild-type (WT) and mutant (harboring Y81C) I(Ks) channels are expressed in oocytes or COS-7 cells. Channel function and KCNQ1 protein expression/subcellular distribution are studied by techniques of electrophysiology, biochemistry, and immunocytochemistry. Ab initio structure predictions of KCNE1 cytoplasmic domain are performed by the Robetta server. Relative to WT KCNE1, Y81C reduces I(Ks) current amplitude and shifts the voltage range of activation to a more positive range. Y81C does not reduce whole-cell KCNQ1 protein level or interfere with KCNQ1 trafficking to cell surface. Thus, its effects are mediated by altered KCNQ1/KCNE1 interactions in cell surface channels. Importantly, Y81C potentiates the effects of an I(Ks) activator. Preserving the aromatic or hydroxyl side chain at position 81 (Y81F or Y81T) does not prevent the detrimental effects of Y81C. Structure predictions suggest that the post-TMD region of KCNE1 may adopt a helical secondary structure. We propose that the post-TMD region of KCNE1 interacts with the KCNQ1 channel to modulate I(Ks) current amplitude and gating kinetics. Other LQT5 mutations in this region share the Y81C phenotype and probably affect the I(Ks) channel function by a similar mechanism.

Dual roles of the A kinase-anchoring protein Yotiao in the modulation of a cardiac potassium channel: A passive adaptor versus an active regulator

Cardiac function is regulated critically by the autonomic nervous system to adapt to the physical activity and emotional stress. A slowly activating cardiac potassium channel ( I Ks) is modulated by stimulation of the sympathetic nervous system (SNS) and contributes to cardiac action potential shortening in the face of concomitant increases in heart rate. Activation of β-adrenergic receptors in response to SNS stimulation results in protein kinase A (PKA)-mediated phosphorylation of I Ks channels. We have found that the functional regulation of the I Ks channel by PKA requires the A kinase-anchoring protein (AKAP) Yotiao. Yotiao forms a macromolecular complex with the channel and recruits key enzymes such as PKA and protein phosphatase 1 (PP1) to control the phosphorylation state of I Ks. Our recent findings revealed a more active role of Yotiao in the PKA modulation of I Ks. We found that Yotiao participates actively in translating the phosphorylation-induced change into altered channel activity. Moreover Yotiao itself can be phosphorylated by PKA upon β-adrenergic stimulation. Ablation of Yotiao phosphorylation impairs PKA-induced changes in I Ks voltage-dependent activation and current kinetics. Taken together we have evidence to suggest that Yotiao plays dual roles in the PKA modulation of the I Ks channel. It acts not only as an adaptor protein to coordinate enzymatic reactions but also as an active regulator that directly affects channel function.