IK Channels Research Articles

Evidence that two distinct crypt cell types secrete chloride and potassium in human colon

BackgroundHuman colon may secrete substantial amounts of water secondary to chloride (Cl−) and/or potassium (K+) secretion in a variety of diarrhoeal diseases. Ion secretion occurs via Cl− and K+ channels,...

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β1‐Adrenoceptor stimulation suppresses endothelial IKCa‐channel hyperpolarization and associated dilatation in resistance arteries

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²⁺-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]i . The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Endothelial caveolar modulation of SK3 channel activity

Small‐ and intermediate‐conductance Ca2+‐activated K+ channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of [Ca2+]i required for the production of endothelium‐derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca2+ influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca2+‐dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca2+‐dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo‐ or endocytosis is block. Thus, modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca2+‐dependent manner.

Distinct potentials in the myoendothelial junction

Myoendothelial junctions (MEJ) bridge the smooth muscle cells (SMCs) and endothelial cells (ECs) in small arteries. In most vessels they project from the EC through the internal elastic lamina and into the SMC, where gap junctions connect the MEJ with the SMC. MEJs are characterised by a long thin neck and on top a “head”, which can be coupled to SMCs with gap junctions. We have used a spatiotemporal model to simulate electrical coupling between the two cell types. The model shows that the MEJ could be electrically isolated from both the EC and the SMC due to the long thin neck and that there could be levels of cations and anions in the MEJ that are different from the EC and SMC bulk levels. Local Ca2+ domains in the MEJ can activate an outward K+ current in the MEJ and we found that activation of less than 10–20 K+ channels (e.g. IK channels) is sufficient to create an electrical potential of 1–5 mV between the SMC and MEJ. This suggests that the MEJ should be considered as a unique structure with properties distinct from those of the smooth muscle cells or of the endothelial cells proper. Supported by The Danish Council for Independent Research ‐ Medical Sciences.

Impaired vasodilation of human obese visceral resistance arteries is improved by apocynin through hydrogen peroxide and activation of SK and IK channels

Previous studies indicate that in human obesity, visceral adipose tissue (VAT) is associated with endothelial dysfunction compared to subcutaneous adipose tissue (SAT). We hypothesized that inhibition of NADPH oxidase will reverse impaired Flow‐induced dilation (FID) in resistance arteries of VAT. Arterioles were dissected from VAT and SAT biopsies (BMI: 48±2, n=15) and cannulated for measurements of luminal diameters. Maximum dilations to papaverine (10−4 M) were determined. Production of hydrogen peroxide (H2O2) was measured by fluorescence microscopy. FID was reduced in VAT to 38±4% (p&lt;0.01, at 60 cm H2O pressure gradient) compared to SAT (92±4%). Apocynin restored FID (73±5%) in VAT and had no effect in SAT. PEG‐catalase abolished the restored FID in the presence of apocynin. Incubation with apamin and tram‐34, blockers of small (SK)‐ and intermediate (IK)‐ conductance, Ca2+‐activated potassium channels, respectively, decreased this restored FID. Improved FID induced by the SK channel opener CyPPA was blocked by apamin. Flow‐ induced H2O2 generation was increased (1.7 fold) in the presence of apocynin compared to baseline and PEG‐catalase attenuated this increase. Taken together these data suggest that H2O2 and the activation of SK and IK channels contribute to the restored effect of apocynin on flow induced dilation in visceral fat during obesity. NIH (R01HL095701).

Lisinopril augments the combined effect of nitric oxide and potassium channels on endothelium‐dependent relaxation in spontaneously hypertensive rats

Endothelium‐dependent relaxation is reported to be impaired in hypertension. However, there is equivocal evidence on the exact nature of the underlying dysfunction and whether it could be affected by antihypertensive drugs specifically angiotensin‐converting enzyme inhibitors. This study was conducted to evaluate the contribution of NO and potassium channels to endothelial function after lisinopril treatment in spontaneously hypertensive rats. Male animals (10–12 weeks old) were treated with lisinopril (20mg/Kg/day) for 10 weeks. ACh (0.1 nM–10μM)‐induced relaxation of third order mesenteric arteries was measured on a wire myograph with isometric recording in the presence and absence of different blockers. In the presence of inhibitor of nitric oxide synthase (LNMMA 100uM), the maximum relaxation of arteries from untreated (UT) and treated (T) rats to ACh was reduced by 19±3.5% and 5.0±01.6% respectively, while in the presence of SK and IK channel blockers (apamin 100nM and TRAM‐34 10μM) it was reduced by 35±3.5% and 34%±6.25%; respectively (n=10–22). With combined blockade, ACh‐induced relaxation was inhibited by 29±6.2% UT and 65±3.2 % T. Our results suggest that chronic treatment with lisinopril, although it reduced the relative contribution of nitric oxide alone it enhanced the combined effect of nitric oxide and potassium channels towards more endothelium‐dependent relaxation.

Human embryonic stem cell derived cardiac myocytes detect hERG-mediated repolarization effects, but not Nav1.5 induced depolarization delay

IntroductionCardiac safety is of paramount importance in contemporary drug development. Efficient and sensitive evaluation of cardiac safety in the research and development of new molecular agents begins with preclinical in-vitro models. A new model that is currently under evaluation is the human embryonic stem-cell derived cardiac myocytes (hESC-CM) (Peng, Lacerda, Kirsch, Brown, & Bruening-Wright, 2010). MethodshESC-CM were exposed in-vitro to 15 test compounds, and action potentials (AP) recorded with perforated patch-clamp technique to assess changes in AP duration (APD90) and upstroke velocity (Vmax). The test compounds included: 10 hERG channel, 4 Na+ channel, and 1 IKs channel inhibitors. For comparison purposes, the test compounds were evaluated in the isolated rabbit heart assay (IRH) to determine changes in conduction (QRS) and repolarization (QTc). Potency at hERG, NaV1.5 and IKs channel was also determined. ResultsFor 7 of 10 hERG channel inhibitors, the potency values across the three functional assays were similar (≤5-fold). Three compounds (dofetilide, sertindole, and terfenadine) showed >10-fold discrepancy between hERG potency and inhibitory concentrations in the hESC-CM and IRH assays. Of the four Na+ channel inhibitors, only mexiletine exhibited similar potency values across the three assays (~3-fold); the others exhibited marked variation (>10-fold) in inhibitory potency. No effect on repolarization was observed in hESC-CM treated with a potent IKs blocker, but QTc prolongation was evident in the IRH. DiscussionThe functional data indicate that hESC-CM are sensitive for detecting repolarization delay induced by hERG channel blockade, and AP prolongation correlated with potency in the hERG channel and IRH assays. However, hESC-CM were less sensitive for detecting depolarizing delay by Na+ channel blockers, and unable to detect delayed repolarization caused by IKs blockade.

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca(2+) -activated K(+) (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity.

Purinergic regulation of CFTR and Ca2+-activated Cl− channels and K+ channels in human pancreatic duct epithelium

Purinergic agonists have been considered for the treatment of respiratory epithelia in cystic fibrosis (CF) patients. The pancreas, one of the most seriously affected organs in CF, expresses various purinergic receptors. Studies on the rodent pancreas show that purinergic signaling regulates pancreatic secretion. In the present study we aim to identify Cl(-) and K(+) channels in human pancreatic ducts and their regulation by purinergic receptors. Human pancreatic duct epithelia formed by Capan-1 or CFPAC-1 cells were studied in open-circuit Ussing chambers. In Capan-1 cells, ATP/UTP effects were dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3.1). The apical effects of ATP/UTP were greatly potentiated by the IK channel opener DC-EBIO. Determination of RNA and protein levels revealed that Capan-1 cells have high expression of TMEM16A (ANO1), a likely CaCC candidate. We conclude that in human pancreatic duct cells ATP/UTP regulates via purinergic receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct function should consider sidedness of purinergic signaling and the essential role of K(+) channels.

Dysfunctional potassium channel subunit interaction as a novel mechanism of long QT syndrome.

The slowly-activating delayed rectifier current IKs contributes to repolarization of the cardiac action potential, and is composed of a pore-forming α-subunit, KCNQ1, and a modulatory β-subunit, KCNE1. Mutations in either subunit can cause long QT syndrome, a potentially fatal arrhythmic disorder. How KCNE1 exerts its extensive control over the kinetics of IKs remains unresolved To evaluate the impact of a novel KCNQ1 mutation on IKs channel gating and kinetics KCNQ1 mutations were expressed in Xenopus oocytes in the presence and absence of KCNE1. Voltage clamping and MODELLER software were used to characterize and model channel function. Mutant and wt genes were cloned into FLAG, Myc and HA expression vectors to achieve differential epitope tagging, and expressed in HEK293 cells for immunohistochemical localization and surface biotinylation assay. We identified 2 adjacent mutations, S338F and F339S, in the KCNQ1 S6 domain in unrelated probands. The novel KCNQ1 S338F mutation segregated with prolonged QT interval and torsade de pointes; the second variant, F339S, was associated with fetal bradycardia and prolonged QT interval, but no other clinical events. S338F channels expressed in Xenopus oocytes had slightly increased peak conductance relative to wild type, with a more positive activation voltage. F339S channels conducted minimal current. Unexpectedly, S338F currents were abolished by co-expression with intact WT KCNE1 or its C-terminus (aa63-129), despite normal membrane trafficking and surface co-localization of KCNQ1 S338F and wt KCNE1. Structural modeling indicated that the S338F mutation specifically alters the interaction between the S6 domain of one KCNQ1 subunit and the S4-S5 linker of another, inhibiting voltage-induced movement synergistically with KCNE1 binding. A novel KCNQ1 mutation specifically impaired channel function in the presence of KCNE1. Our structural model shows that this mutation effectively immobilizes voltage gating by an inhibitory interaction that is additive with that of KCNE1. Our findings illuminate a previously unreported mechanism for LQTS, and validate recent theoretical models of the closed state of the KCNQ1:KCNE1 complex.

Binding of ATP is Required for Opening of IKs Channels

The human heart demands a constant energy supply to fulfill its function, hence the decrease in myocardial ATP production plays a key role in the pathogenesis and progression of ischemic heart diseases. Ischemia also alters electrophysiology of the heart, exemplified by an association between an adverse prognosis and QT interval prolongation in acute myocardial ischemia. KCNQ1 and KCNE1 form the IKs potassium channel important in terminating cardiac action potentials. Congenital mutations that compromise IKs function prolong the duration of the ventricular action potential, causing Long QT (LQT) syndrome, which is associated with a high risk of sudden death. Here we report that the IKs channel activity increases with ATP concentration ([ATP]) and the EC50 is close to the physiological [ATP] in cardiac myocytes, which indicates that [ATP] changes such as in ischemia affect IKs channel function. Consistent with this observation, an LQT-associated mutation in KCNQ1 alters IKs function by reducing ATP sensitivity. The effect of this mutation is eliminated by increasing [ATP]. We find that GTP and a non-hydrolyzable ATP analog AMP-PNP can substitute for ATP in activating the channel, and an ATP analog can be photo-cross-linked to KCNQ1 proteins expressed in the membrane of Xenopus oocytes, indicating that the nucleotide directly binds to KCNQ1 to modify channel function. Compared to ATP, ADP and AMP are less effective in activating the IKs channel, suggesting that phosphate groups are important innucleotide binding. Correspondingly, a mutational scan of all cytosolic basic residues shows that the ATP binding site may reside in the cytosolic C-terminal. These results demonstrate that IKs is a bona fide ATP activated potassium channel that modulates cardiac electrophysiology by sensing intracellular [ATP], thus connecting the cellular energy state to membrane excitability.

The Mechanism by which ATP Binding Opens IKs Channels

In cardiac myocytes KCNQ1 and KCNE1 form the IKs channel that is important in terminating action potentials. KCNQ1 has a common Kv channel membrane topology with the central pore domain (PD, S5-S6) and the voltage-sensing domain (VSD, S1-S4). We have demonstrated that ATP directly binds to the cytosolic C-terminus of KCNQ1 to regulate IKs channel function. Here we study if ATP regulates VSD movements or pore opening. using inside-out patch clamp techniques, we found that the steady state G-V relationship and current kinetics of the WT IKs channel are not changed by [ATP]. Consistent with these results, mutations in the putative ATP binding site reduce ATP sensitivity of the current amplitude but do not alter G-V relationship or activation kinetics. Furthermore, using voltage clamp fluorometry, we measured ionic current and VSD movements of the WT and mutant channels in which ATP binding is eliminated; and our results show that florescence signals remain the same, while the ionic current is completely absent in the mutant channels due to the lack of ATP binding. These results suggest that ATP does not regulate VSD movements but is required for pore opening by directly pulling the activation gate to open from the binding site located downstream from S6. Supporting this mechanism, a mutation located between S6 and the putative ATP binding site is found to alter the coupling between ATP binding and channel opening by reducing ATP sensitivity and shifting the G-V relationship to more positive voltages at a given [ATP]; however, increasing [ATP] shifts the G-V back toward negative voltages. The effects of this mutation can be explained by an allosteric model for the coupling between ATP binding and channel opening in which the mutation reduces the allosteric factor for the coupling.

Regulation of Voltage Sensor Movement in KCNQ Channels by KCNE Beta Subunits

The KCNQ1 potassium channel plays widely different physiological roles different cell types. In the heart KCNQ1 forms part of the voltage-gated IKs channels that limit the duration of the cardiac action potential, whereas in epithelial cells KCNQ1 forms part of a voltage-independent K+ channel that is important for ion secretion. The different functions of KCNQ1 are mainly due to the co-assembly of KCNQ1 with different KCNE beta subunits. For example, the IKS channel consists of 4 α-subunits (KCNQ1) which assemble with 2 to 4 β subunits (KCNE1), whereas in epithelial cells KCNQ1 co-assembles with KCNE3 to form voltage-independent K+ channels. Mutations in either KCNQ1 or KCNE1 cause cardiac arrhythmia syndromes. Here we use Voltage clamp fluorometry (VCF) to directly study the effects of wild type and mutant KCNE subunits on the voltage sensor movement in KCNQ1 channel. We assess the voltage sensor movement (fluorescence) and channel opening (current), in order to understand the coupling between the KCNQ1 voltage sensor and channel gate in the presence of KCNE subunits. Our data shows that KCNE1 splits the voltage sensor movement in two separates phases: one at hyperpolarized potentials that moves the voltage sensor to an active state and a second at more depolarized potentials, which is tightly coupled to channel opening. Interestingly, VCF shows that KCNE3 locks the voltage sensor of KCNQ1, presumably in its activated state, thereby generating a voltage-independent K+ channel. Furthermore, arrhythmia-causing mutations in KCNE1 shift the voltage dependence of the two different voltage sensor movements, revealing some of the molecular mechanisms underlying these arrhythmia-causing mutations. Our data suggests a putative mechanism for how KCNE1 subunit exerts its effects on the voltage sensor movement during IKs channel activation.

Long QT Mutations and KCNE1 β-Subunit Modulate S4 Movement in KCNQ1 Channel

The KCNQ1 potassium channel is expressed in many different cell types and plays widely different physiological roles. For example, in the heart KCNQ1 forms part of the voltage-gated IKs channels that contributes to limiting the duration of the cardiac action potential. The different functions of the KCNQ1 channel are mainly due to the co-assembly of the KCNQ1 channel with different beta subunits from the KCNE family. The IKS channel consists of 4 α-subunits (KCNQ1) which assemble with 2 to 4 β subunits (KCNE1). Mutations in either KCNQ1 or KCNE1 subunit cause multiple cardiac arrhythmia syndromes such as long QT syndrome, short QT syndrome, and familial atrial fibrillation. Here we use Voltage clamp fluorometry (VCF) to directly study the effects of wild type, mutant KCNQ1 and KCNE1 subunits on the voltage sensor movement in KCNQ1 channel. We assess the voltage sensor movement (fluorescence) and channel opening (current), in order to understand the coupling between the KCNQ1 voltage sensor and channel gate in the presence of KCNE1. Our data show that KCNE1 splits the voltage sensor movement in two separates phases. An early phase (1) involving voltage sensor movements to its active state upon changes in the membrane potential occurs at hyperpolarized potentials. A second phase (2) that happens at much more depolarized potentials involves an additional voltage sensor movement which is tightly coupled to channel opening. Mutations in KCNE1 that cause arrhythmia shift the voltage dependence of the voltage sensor movements of either phases 1 or 2 (or both), revealing some of the molecular mechanisms underlying the pathophysiology of these arrhythmia-causing mutations. Our data suggests a putative mechanism for how KCNE1 exerts its effects on the voltage sensor movement during IKs channel activation.

The Distal Kcne1 C-Terminus is Crucial for Yotiao Mediated Pka-Dependent Phosphorylation of KCNQ1

The assembly of KCNQ1 with KCNE1 produces the IKSpotassium current that is crucial for the late repolarization of the cardiac action potential. Mutations in either KCNQ1 or KCNE1 genes produce the long QT or short QT syndromes, which are genetically heterogeneous cardiovascular diseases, characterized by ventricular or atrial arrhythmias. The scaffolding A-kinase anchoring protein (AKAP) Yotiao brings together PKA, PP1, PDE4D3, AC9, and the IKS channel complex to achieve regulation following beta adrenergic stimulation.We recently showed that the distal KCNE1 C-terminus interacts with the coiled-coil helix C of KCNQ1 C-terminus. Here we examined the effect of LQT mutations located at this C-terminal interface of the two subunits. Four KCNQ1 LQT mutations located at helix C, S546L, K557E, R555C, and R562M impaired the interaction with KCNE1 C-terminus and produced a drastic reduction in IKS current density mostly caused by a right-shift of the voltage-dependence of channel activation. A much weaker PIP2 binding paralleled the decrease in IKS current density. The KCNE1 LQT mutation, P127T, situated at the distal C-terminus weakened the interaction with KCNQ1 helix C and caused a 40% decrease in IKS current density but with no shift of the voltage-dependence of channel activation. Interestingly, the KCNE1 mutant P127T markedly reduced the cAMP-dependent Yotiao-mediated upregulation of IKS current.While the P127T mutation did not alter the ability of KCNQ1 to interact with Yotiao, it strongly disrupted KCNQ1 phosphorylation of S27, in the presence of Yotiao and cAMP. Similar results were found with a deletion of the distal KCNE1 C-terminus.(del 109-129). These results suggest that the distal KCNE1 C-terminus, probably via its interaction with the coiled-coil helix C, is a crucial determinant for the functional modulation of KCNQ1 by Yotiao-mediated PKA phosphorylation.

SKA-31, a novel activator of SKCa and IKCa channels, increases coronary flow in male and female rat hearts

Endothelial SK(Ca) and IK(Ca) channels play an important role in the regulation of vascular function and systemic blood pressure. Based on our previous findings that small molecule activators of SK(Ca) and IK(Ca) channels (i.e. NS309 and SKA-31) can inhibit myogenic tone in isolated resistance arteries, we hypothesized that this class of compounds may induce effective vasodilation in an intact vascular bed, such as the coronary circulation. In a Langendorff-perfused, beating rat heart preparation, acute bolus administrations of SKA-31 (0.01-5 µg) dose-dependently increased total coronary flow (25-30%) in both male and female hearts; these responses were associated with modest, secondary increases in left ventricular (LV) systolic pressure and heart rate. SKA-31 evoked responses in coronary flow, LV pressure, and heart rate were qualitatively comparable to acute responses evoked by bradykinin (1 µg) and adenosine (10 µg). In the presence of apamin and TRAM-34, selective blockers of SK(Ca) and IK(Ca) channels, respectively, SKA-31 and bradykinin-induced responses were largely inhibited, whereas the adenosine-induced changes were blocked by ∼40%; TRAM-34 alone produced less inhibition. Sodium nitroprusside (SNP, 0.2 μg bolus dose) evoked changes in coronary flow, LV pressure, and heart rate were similar to those induced by SKA-31, but were unaffected by apamin + TRAM-34. The NOS inhibitor L-NNA reduced bradykinin- and adenosine-evoked changes, but did not affect responses to either SKA-31 or SNP. Our study demonstrates that SKA-31 can rapidly and reversibly induce dilation of the coronary circulation in intact functioning hearts under basal flow and contractility conditions.

Differential role of IK and BK potassium channels as mediators of intrinsic and extrinsic apoptotic cell death

An important event during apoptosis is regulated cell condensation known as apoptotic volume decrease (AVD). Ion channels have emerged as essential regulators of this process mediating the release of K(+) and Cl(-), which together with osmotically obliged water, results in the condensation of cell volume. Using a Grade IV human glioblastoma cell line, we examined the contribution of calcium-activated K(+) channels (K(Ca) channels) to AVD after the addition of either staurosporine (Stsp) or TNF-α-related apoptosis-inducing ligand (TRAIL) to activate the intrinsic or extrinsic pathway of apoptosis, respectively. We show that AVD can be inhibited in both pathways by high extracellular K(+) or the removal of calcium. However, BAPTA-AM was only able to inhibit Stsp-initiated AVD, whereas TRAIL-induced AVD was unaffected. Specific K(Ca) channel inhibitors revealed that Stsp-induced AVD was dependent on K(+) efflux through intermediate-conductance calcium-activated potassium (IK) channels, while TRAIL-induced AVD was mediated by large-conductance calcium-activated potassium (BK) channels. Fura-2 imaging demonstrated that Stsp induced a rapid and modest rise in calcium that was sustained over the course of AVD, while TRAIL produced no detectable rise in global intracellular calcium. Inhibition of IK channels with clotrimazole or 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) blocked downstream caspase-3 activation after Stsp addition, while paxilline, a specific BK channel inhibitor, had no effect. Treatment with ionomycin also induced an IK-dependent cell volume decrease. Together these results show that calcium is both necessary and sufficient to achieve volume decrease and that the two major pathways of apoptosis use unique calcium signaling to efflux K(+) through different K(Ca) channels.

Repolarization reserve determines drug responses in human pluripotent stem cell derived cardiomyocytes

Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca2+ channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected.Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (IKs), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional IKs channels. These compounds were therefore re-evaluated under conditions that mimicked reduced “repolarization reserve,” a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that IKs plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety.