-
[show abstract]
[hide abstract]
ABSTRACT: Lone atrial fibrillation (AF) is associated with various ion channel gene sequence variants, notably the common S38G loss-of-function polymorphism in the KCNE1 K(+) channel ancillary subunit gene. New-onset postoperative AF (POAF) generally occurs 48-72 h after major surgery, particularly following procedures within the chest, but its molecular bases remain poorly understood. To begin to address this gap in knowledge, we analyzed molecular changes in the left atrium (LA) in relation to simultaneous changes in hemodynamics, LA effective refractory period (ERP), and the capacity to sustain electrically-induced AF following left upper lung lobectomy in swine. Relative to control pigs (no previous surgery), 3 days after lobectomy higher values for mean pulmonary artery pressure (16 ± 1 vs 22 ± 2 mmHg; P=0.045) and pulmonary vascular resistance (273 ± 47 vs 481 ± 63 dyns/cm(5); P=0.025) were evident, whereas other hemodynamic variables were unchanged. LA ERP trended toward reduction in lobectomy animals (187 ± 16 vs 170 ± 20 ms, P>0.05). None of the lobectomy pigs developed spontaneous POAF as assessed by telemetric ECG. However, all lobectomy pigs, but none of the controls, were able to sustain AF induced by a 10s burst of rapid pacing for ≥ 30 s (P=0.0079), independent of LA ERP; AF was sustained ≥ 60s in 3/5 postoperative pigs versus 0/5 controls and correlated with a shorter ERP overall (P=0.023). Transcriptomic analysis of LA tissue revealed 23 up-regulated and 10 down-regulated transcripts (≥ 1.5-fold, P<0.05) in lobectomy pigs. Strikingly, of the latter, KCNE1 down-regulation showed the statistically strongest link to surgery (2.0-fold, P=0.009), recapitulated at the protein level with Western blotting (P=0.039), suggesting KCNE1 down-regulation as a possible common mechanistic factor in POAF and lone AF. Of the up-regulated transcripts, while Teneurin-2 was the strongest linked (1.5-fold change, P=0.001), DSCR5 showed the highest induction (2.7-fold, P=0.02); this and other hits will be targeted in future functional studies.
Journal of Molecular and Cellular Cardiology 05/2012; 53(3):350-3. · 5.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The KCNQ1 α subunit and the KCNE2 β subunit form a potassium channel in thyroid epithelial cells. Genetic disruption of KCNQ1-KCNE2 causes hypothyroidism in mice, resulting in cardiac hypertrophy, dwarfism, alopecia, and prenatal mortality. Here, we investigated the mechanistic requirement for KCNQ1-KCNE2 in thyroid hormone biosynthesis, utilizing whole-animal dynamic positron emission tomography. The KCNQ1-specific antagonist (-)-[3R,4S]-chromanol 293B (C293B) significantly impaired thyroid cell I(-) uptake, which is mediated by the Na(+)/I(-) symporter (NIS), in vivo (dSUV/dt: vehicle, 0.028 ± 0.004 min(-1); 10 mg/kg C293B, 0.009 ± 0.006 min(-1)) and in vitro (EC(50): 99 ± 10 μM C293B). Na(+)-dependent nicotinate uptake by SMCT, however, was unaffected. Kcne2 deletion did not alter the balance of free vs. thyroglobulin-bound I(-) in the thyroid (distinguished using ClO(4)(-), a competitive inhibitor of NIS), indicating that KCNQ1-KCNE2 is not required for Duox/TPO-mediated I(-) organification. However, Kcne2 deletion doubled the rate of free I(-) efflux from the thyroid following ClO(4)(-) injection, a NIS-independent process. Thus, KCNQ1-KCNE2 is necessary for adequate thyroid cell I(-) uptake, the most likely explanation being that it is prerequisite for adequate NIS activity.
The FASEB Journal 05/2012; 26(8):3252-9. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Atrial fibrillation, a common cardiac arrhythmia, often progresses unfavourably: in patients with long-term atrial fibrillation, fibrillatory episodes are typically of increased duration and frequency of occurrence relative to healthy controls. This is due to electrical, structural, and contractile remodeling processes. We investigated mechanisms of how electrical and structural remodeling contribute to perpetuation of simulated atrial fibrillation, using a mathematical model of the human atrial action potential incorporated into an anatomically realistic three-dimensional structural model of the human atria. Electrical and structural remodeling both shortened the atrial wavelength--electrical remodeling primarily through a decrease in action potential duration, while structural remodeling primarily slowed conduction. The decrease in wavelength correlates with an increase in the average duration of atrial fibrillation/flutter episodes. The dependence of reentry duration on wavelength was the same for electrical vs. structural remodeling. However, the dynamics during atrial reentry varied between electrical, structural, and combined electrical and structural remodeling in several ways, including: (i) with structural remodeling there were more occurrences of fragmented wavefronts and hence more filaments than during electrical remodeling; (ii) dominant waves anchored around different anatomical obstacles in electrical vs. structural remodeling; (iii) dominant waves were often not anchored in combined electrical and structural remodeling. We conclude that, in simulated atrial fibrillation, the wavelength dependence of reentry duration is similar for electrical and structural remodeling, despite major differences in overall dynamics, including maximal number of filaments, wave fragmentation, restitution properties, and whether dominant waves are anchored to anatomical obstacles or spiralling freely.
PLoS Computational Biology 02/2012; 8(2):e1002390. · 5.22 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated potassium (Kv) channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and non-excitable cells. With 40 known members in the human genome and a variety of homomeric and heteromeric pore-forming α subunit interactions, post-translational modifications, cellular locations, and expression patterns, the functional repertoire of the Kv α subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv α subunit partners.
Frontiers in physiology. 01/2012; 3:231.
-
[show abstract]
[hide abstract]
ABSTRACT: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I(h), which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.
We investigated the effects of Kcne2 gene deletion on I(h) properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2(+/+) and Kcne2(-/-) mice. Kcne2 deletion shifted the voltage-dependence of I(h) activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I(h) density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2(-/-) neurons.
Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.
PLoS ONE 01/2012; 7(8):e42756. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation-a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.
Biophysical Journal 09/2011; 101(6):1354-63. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated potassium (Kv) currents generated by N-type α-subunit homotetramers inactivate rapidly because an N-terminal ball domain blocks the channel pore after activation. Hence, the inactivation rate of heterotetrameric channels comprising both N-type and non-N-type (delayed rectifier) α-subunits depends upon the number of N-type α-subunits in the complex. As Kv channel inactivation and inactivation recovery rates regulate cellular excitability, the composition and expression of these heterotetrameric complexes are expected to be tightly regulated. In a companion article, we showed that the single transmembrane segment ancillary (β) subunits KCNE1 and KCNE2 suppress currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels, by trapping them early in the secretory pathway. Here, we show that this trapping is prevented by coassembly of the N-type α-subunits with intra-subfamily delayed rectifier α-subunits. Extra-subfamily delayed rectifier α-subunits, regardless of their capacity to interact with KCNE1 and KCNE2, cannot rescue Kv1.4 or Kv3.4 surface expression unless engineered to interact with them using N-terminal A and B domain swapping. The KCNE1/2-enforced checkpoint ensures N-type α-subunits only reach the cell surface as part of intra-subfamily mixed-α complexes, thereby governing channel composition, inactivation rate, and-by extension-cellular excitability.
Biophysical Journal 09/2011; 101(6):1364-75. · 3.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cerebrospinal fluid (CSF) is crucial for normal function and mechanical protection of the CNS. The choroid plexus epithelium (CPe) is primarily responsible for secreting CSF and regulating its composition by mechanisms currently not fully understood. Previously, the heteromeric KCNQ1-KCNE2 K(+) channel was functionally linked to epithelial processes including gastric acid secretion and thyroid hormone biosynthesis. Here, using Kcne2(-/-) tissue as a negative control, we found cerebral expression of KCNE2 to be markedly enriched in the CPe apical membrane, where we also discovered expression of KCNQ1. Targeted Kcne2 gene deletion in C57B6 mice increased CPe outward K(+) current 2-fold. The Kcne2 deletion-enhanced portion of the current was inhibited by XE991 (10 μM) and margatoxin (10 μM) but not by dendrotoxin (100 nM), indicating that it arose from augmentation of KCNQ subfamily and KCNA3 but not KCNA1 K(+) channel activity. Kcne2 deletion in C57B6 mice also altered the polarity of CPe KCNQ1 and KCNA3 trafficking, hyperpolarized the CPe membrane by 9 ± 2 mV, and increased CSF [Cl(-)] by 14% compared with wild-type mice. These findings constitute the first report of CPe dysfunction caused by cation channel gene disruption and suggest that KCNE2 influences blood-CSF anion flux by regulating KCNQ1 and KCNA3 in the CPe.
The FASEB Journal 08/2011; 25(12):4264-73. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The slow-activating cardiac repolarization K(+) current (I(Ks)), generated by the KCNQ1-KCNE1 potassium channel complex, is controlled via sympathetic and parasympathetic regulation in vivo. Inherited KCNQ1 and KCNE1 mutations predispose to ventricular fibrillation and sudden death, often triggered by exercise or emotional stress. Protein kinase C (PKC), which is activated by α1 adrenergic receptor stimulation, is known to downregulate I(Ks) via phosphorylation of KCNE1 serine 102, but the underlying mechanism has remained enigmatic. We previously showed that KCNE1 mediates dynamin-dependent endocytosis of KCNQ1-KCNE1 complexes.
This study sought to determine the potential role of endocytosis in I(Ks) downregulation by PKC.
We utilized patch clamping and fluorescence microscopy to study Chinese hamster ovary (CHO) cells coexpressing KCNQ1, KCNE1, and wild-type or dominant-negative mutant (K44A) dynamin 2, and neonatal mouse ventricular myocytes.
The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased I(Ks) density by >60% (P < .05) when coexpressed with wild-type dynamin 2 in CHO cells, but had no effect when coexpressed with K44A-dynamin 2. Thus, functional dynamin was required for downregulation of I(Ks) by PKC activation. PMA increased KCNQ1-KCNE1 endocytosis in CHO cells expressing wild-type dynamin 2, but had no effect on KCNQ1-KCNE1 endocytosis in CHO cells expressing K44A-dynamin 2, determined using the Pearson correlation coefficient to quantify endosomal colocalization of KCNQ1 and KCNE1 with internalized fluorescent transferrin. KCNE1-S102A abolished the effect of PMA on I(Ks) currents and endocytosis. Importantly, PMA similarly stimulated endocytosis of endogenous KCNQ1 and KCNE1 in neonatal mouse myocytes.
PKC activation downregulates I(Ks) by stimulating KCNQ1-KCNE1 channel endocytosis.
Heart rhythm: the official journal of the Heart Rhythm Society 05/2011; 8(10):1641-7. · 4.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Targeted deletion of the Kcne2 potassium channel β subunit gene ablates gastric acid secretion and predisposes to gastric neoplasia in mice. Here, we discovered that Kcne2 deletion basolaterally reroutes the Kcnq1 α subunit in vivo in parietal cells (PCs), in which the normally apical location of the Kcnq1-Kcne2 channel facilitates its essential role in gastric acid secretion. Quantitative RT-PCR and Western blotting revealed that Kcne2 deletion remodeled fundic Kcne3 (2.9±0.8-fold mRNA increase, n=10; 5.3±0.4-fold protein increase, n=7) but not Kcne1, 4, or 5, and resulted in basolateral Kcnq1-Kcne3 complex formation in Kcne2(-/-) PCs. Concomitant targeted deletion of Kcne3 (creating Kcne2(-/-)Kcne3(-/-) mice) restored PC apical Kcnq1 localization without Kcne1, 4, or 5 remodeling (assessed by quantitative RT-PCR; n=5-10), indicating Kcne3 actively, basolaterally rerouted Kcnq1 in Kcne2(-/-) PCs. Despite this, Kcne3 deletion exacerbated gastric hyperplasia in Kcne2(-/-) mice, and both hypochlorhydria and hyperplasia in Kcne2(+/-) mice, suggesting that Kcne3 up-regulation was beneficial in Kcne2-depleted PCs. The findings reveal, in vivo, Kcne-dependent α subunit polarized trafficking and the existence and consequences of potassium channel β subunit remodeling.
The FASEB Journal 11/2010; 25(2):727-36. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Inherited Long QT Syndrome (LQTS), a cardiac arrhythmia that predisposes to the often lethal ventricular fibrillation, is commonly linked to mutations in KCNQ1. The KCNQ1 voltage-gated K(+) channel α subunit passes ventricular myocyte K(+) current that helps bring a timely end to each heart-beat. KCNQ1, like many K(+) channel α subunits, is regulated by KCNE β subunits, inherited mutations in which also associate with LQTS. KCNQ1 and KCNE mutations are also associated with atrial fibrillation. It has long been known that thyroid status strongly influences cardiac function, and that thyroid dysfunction causes abnormal cardiac structure and rhythm. We recently discovered that KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated K(+) channel in the thyroid that is required for normal thyroid hormone biosynthesis. Here, we review this novel genetic link between cardiac and thyroid physiology and pathology, and its potential influence upon future therapeutic strategies in cardiac and thyroid disease.
The international journal of biochemistry & cell biology 11/2010; 42(11):1767-70. · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Continuous, rhythmic beating of the heart requires exquisite control of expression, localization and function of cardiac ion channels - the foundations of the cardiac myocyte action potential. Disruption of any of these processes can alter the shape of the action potential, predisposing to cardiac arrhythmias. These arrhythmias can manifest in a variety of ways depending on both the channels involved and the type of disruption (i.e., gain or loss of function). As much as 1% of the population of developed countries is affected by cardiac arrhythmia each year, and a detailed understanding of the mechanism of each arrhythmia is crucial to developing and prescribing the proper therapies. Many of the antiarrhythmic drugs currently on the market were developed before the underlying cause of the arrhythmia was known, and as a result lack specificity, causing side effects. The majority of the available drugs target the conductance of cardiac ion channels, either by blocking or enhancing current through the channel. In recent years, however, it has become apparent that specific targeting of ion channel conductance may not be the most effective means for treatment. Here we review increasing evidence that suggests defects in ion channel trafficking play an important role in the etiology of arrhythmias, and small molecule approaches to correct trafficking defects will likely play an important role in the future of arrhythmia treatment.
Expert Review of Cardiovascular Therapy 08/2010; 8(8):1161-73.
-
[show abstract]
[hide abstract]
ABSTRACT: The low-dielectric plasma membrane provides an energy barrier hindering transmembrane movement of charged particles. The positively charged, voltage-sensing fourth transmembrane domain (S4) of voltage-gated ion channels must surmount this energy barrier to initiate channel activation, typically necessitating both membrane depolarization and interaction with membrane lipid phospho-head groups (MLPHGs). In contrast, and despite containing S4, the KCNQ1 K(+) channel alpha subunit exhibits predominantly constitutive activation when in complexes with transmembrane beta subunits, MinK-related peptide (MiRP) 1 (KCNE2) or MiRP2 (KCNE3). Here, using a 2-electrode voltage clamp and scanning mutagenesis of channels heterologously expressed in Xenopus laevis oocytes, we discovered that 2 of the 8 MiRP2 extracellular domain acidic residues (D54 and D55) are important for KCNQ1-MiRP2 constitutive activation. Double-mutant thermodynamic cycle analysis revealed energetic coupling of D54 and D55 to R237 in KCNQ1 S4 but not to 10 other native or introduced polar residues in KCNQ1 S4 and surrounding linkers. MiRP2-D54 and KCNQ1-R237 also similarly dictated susceptibility to the inhibitory effects of MLPHG hydrolysis, whereas other closely situated polar residues did not. Thus, by providing negative charge near the plasma membrane extracellular face, MiRP2 uses a lipomimetic mechanism to constitutively stabilize the activated KCNQ1 voltage sensor.
The FASEB Journal 05/2010; 24(5):1518-24. · 5.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 alpha subunit and the KCNE2 beta subunit provide a K(+) efflux current to facilitate gastric acid secretion by the apical H(+)K(+)ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2(-/-) mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2(-/-) mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia.
PLoS ONE 01/2010; 5(7):e11451. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia. Here, we show that the potassium channel subunits KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated, constitutively active, thyrocyte K+ channel required for normal thyroid hormone biosynthesis. Targeted disruption of Kcne2 in mice impaired thyroid iodide accumulation up to eightfold, impaired maternal milk ejection, halved milk tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening. The alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T3) and T4 administration to pups, by supplementing dams with T(4) before and after they gave birth or by feeding the pups exclusively from Kcne2+/+ dams; conversely, these symptoms were elicited in Kcne2+/+ pups by feeding exclusively from Kcne2-/- dams. These data provide a new potential therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1-linked human cardiac arrhythmias.
Nature medicine 09/2009; 15(10):1186-94. · 27.14 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cardiac repolarization alternans is an arrhythmogenic rhythm disturbance, manifested in individual myocytes as a beat-to-beat alternation of action potential durations and intracellular calcium transient magnitudes. Recent experimental studies have reported "subcellular alternans," in which distinct regions of an individual cell are seen to have counterphase calcium alternations, but the mechanism by which this occurs is not well understood. Although previous theoretical work has proposed a possible dynamical mechanism for subcellular alternans formation, no direct evidence for this mechanism has been reported in vitro. Rather, experimental studies have generally invoked fixed subcellular heterogeneities in calcium-cycling characteristics as the mechanism of subcellular alternans formation.
In this study, we have generalized the previously proposed dynamical mechanism to predict a simple pacing algorithm by which subcellular alternans can be induced in isolated cardiac myocytes in the presence or absence of fixed subcellular heterogeneity. We aimed to verify this hypothesis using computational modeling and to confirm it experimentally in isolated cardiac myocytes. Furthermore, we hypothesized that this dynamical mechanism may account for previous reports of subcellular alternans seen in statically paced, intact tissue.
Using a physiologically realistic computational model of a cardiac myocyte, we show that our predicted pacing algorithm induces subcellular alternans in a manner consistent with theoretical predictions. We then use a combination of real-time electrophysiology and fluorescent calcium imaging to implement this protocol experimentally and show that it robustly induces subcellular alternans in isolated guinea pig ventricular myocytes. Finally, we use computational modeling to demonstrate that subcellular alternans can indeed be dynamically induced during static pacing of 1D fibers of myocytes during tissue-level spatially discordant alternans.
Here we provide the first direct experimental evidence that subcellular alternans can be dynamically induced in cardiac myocytes. This proposed mechanism may contribute to subcellular alternans formation in the intact heart.
Circulation Research 08/2009; 105(4):335-42. · 9.49 Impact Factor
-
AJP Heart and Circulatory Physiology 04/2009; 296(5):H1211-2. · 3.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: KCNQ1-MinK potassium channel complexes (4alpha:2beta stoichiometry) generate IKs, the slowly activating human cardiac ventricular repolarization current. The MinK ancillary subunit slows KCNQ1 activation, eliminates its inactivation, and increases its unitary conductance. However, KCNQ1 transcripts outnumber MinK transcripts five to one in human ventricles, suggesting KCNQ1 also forms other heteromeric or even homomeric channels there. Mechanisms governing which channel types prevail have not previously been reported, despite their significance: normal cardiac rhythm requires tight control of IKs density and kinetics, and inherited mutations in KCNQ1 and MinK can cause ventricular fibrillation and sudden death. Here, we describe a novel mechanism for this control.
Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native IKs channels in guinea-pig myocytes. KCNQ1-MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1-MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse.
The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition-and thus current density and gating kinetics-that may also apply to other alpha-beta type Kv channel complexes.
Cardiovascular research 03/2009; 82(3):430-8. · 5.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously showed that activation of G(i/o)-coupled histamine H(3)-receptors (H(3)R) is cardioprotective because it attenuates excessive norepinephrine release from cardiac sympathetic nerves. This action is characterized by a marked decrease in intraneuronal Ca(2+) ([Ca(2+)](i)), as G alpha(i) impairs the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway, and this decreases Ca(2+) influx via voltage-operated Ca(2+) channels (VOCC). Yet, the G(i/o)-derived betagamma dimer could directly inhibit VOCC, and the subsequent reduction in Ca(2+) influx would be responsible for the H(3)R-mediated attenuation of transmitter exocytosis. In this study, we tested this hypothesis in nerve-growth factor-differentiated rat pheochromocytoma cells (PC12) stably transfected with H(3)R (PC12-H(3)) and with the G betagamma scavenger beta-adrenergic receptor kinase 1 (beta-ARK1)-(495-689)-polypeptide (PC12-H(3)/beta-ARK1). Thus, we evaluated the effects of H(3)R activation directly on the following: 1) Ca(2+) current (I(Ca)) using the whole-cell patch-clamp technique; and 2) K(+)-induced exocytosis of endogenous dopamine. H(3)R activation attenuated both peak I(Ca) and dopamine exocytosis in PC12-H(3) but not in PC12-H(3)/beta-ARK1 cells. Moreover, a membrane permeable phosducin-like G betagamma scavenger also prevented the antiexocytotic effect of H(3)R activation. In contrast, the H(3)R-induced attenuation of cAMP accumulation and dopamine exocytosis in response to forskolin were the same in both PC12-H(3) and PC12-H(3)/beta-ARK1 cells. Our findings reveal that although G alpha(i) participates in the H(3)-mediated antiexocytotic effect when the adenylyl cyclase-cAMP-PKA pathway is stimulated, a direct G betagamma-induced inhibition of VOCC, resulting in an attenuation of I(Ca), plays a pivotal role in the H(3)R-mediated decrease in [Ca(2+)](i) and associated cardioprotective antiexocytotic effects. The discovery of this H(3)R-signaling step may offer new therapeutic approaches to cardiovascular diseases characterized by hyperadrenergic activity.
Journal of Pharmacology and Experimental Therapeutics 09/2008; 326(3):871-8. · 3.83 Impact Factor
-
Torsten K Roepke,
Andrianos Kontogeorgis,
Christopher Ovanez,
Xianghua Xu,
Jeffrey B Young,
Kerry Purtell,
Peter A Goldstein,
David J Christini,
Nicholas S Peters,
Fadi G Akar,
David E Gutstein,
Daniel J Lerner, Geoffrey W Abbott
[show abstract]
[hide abstract]
ABSTRACT: Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (-/-) ventricles exhibited a 50% reduction in I(K,slow1), generated by Kv1.5--a previously unknown partner for MiRP1. I(to,f), generated by Kv4 alpha subunits, was also diminished, by approximately 25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (-/-) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K(+) currents and prolonged ventricular APD, kcne2 deletion lengthened the QT(c) under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in alpha subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart.
The FASEB Journal 08/2008; 22(10):3648-60. · 5.71 Impact Factor
