Propofol, an Anesthetic Agent, Inhibits HCN Channels through the Allosteric Modulation of the cAMP-Dependent Gating Mechanism.

Propofol is a broadly used intravenous anesthetic agent that can cause cardiovascular effects, including bradycardia and asystole. A possible mechanism for these effects is slowing cardiac pacemaker activity due to inhibition of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. However, it remains unclear how propofol affects the allosteric nature of the voltage- and cAMP-dependent gating mechanism in HCN channels. To address this aim, we investigated the effect of propofol on HCN channels (HCN4 and HCN2) in heterologous expression systems using a whole-cell patch clamp technique. The extracellular application of propofol substantially suppressed the maximum current at clinical concentrations. This was accompanied by a hyperpolarizing shift in the voltage dependence of channel opening. These effects were significantly attenuated by intracellular loading of cAMP, even after considering the current modification by cAMP in opposite directions. The differential degree of propofol effects in the presence and absence of cAMP was rationalized by an allosteric gating model for HCN channels, where we assumed that propofol affects allosteric couplings between the pore, voltage-sensor, and cyclic nucleotide-binding domain (CNBD). The model predicted that propofol enhanced autoinhibition of pore opening by unliganded CNBD, which was relieved by the activation of CNBD by cAMP. Taken together, these findings reveal that propofol acts as an allosteric modulator of cAMP-dependent gating in HCN channels, which may help us to better understand the clinical action of this anesthetic drug.

Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways.

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both ß1- and ß2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by ß2-adrenoceptor stimulation than ß1-adrenoceptor stimulation. Both ß-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the ß-adrenoceptor-potentiated firing rate. The stimulatory effects of ß- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under ß-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

A hERG mutation E1039X produced a synergistic lesion on IKs together with KCNQ1-R174C mutation in a LQTS family with three compound mutations.

Congenital long QT syndrome (LQTS) caused by compound mutations is usually associated with more severe clinical phenotypes. We identified a LQTS family harboring three compound mutations in different genes (KCNQ1-R174C, hERG-E1039X and SCN5A-E428K). KCNQ1-R174C, hERG-E1039X and SCN5A-E428K mutations and/or relevant wild-type (WT) cDNAs were respectively expressed in mammalian cells. IKs-like, IKr-like, INa-like currents and the functional interaction between KCNQ1-R174C and hERG-E1039X channels were studied using patch-clamp and immunocytochemistry techniques. (1) Expression of KCNQ1-R174C alone showed no IKs. Co-expression of KCNQ1-WT + KCNQ1-R174C caused a loss-of-function in IKs and blunted the activation of IKs in response to isoproterenol. (2) Expression of hERG-E1039X alone and co-expression of hERG-WT + hERG-E1039X negatively shifted inactivation curves and decelerated the recovery time from inactivation. (3) Expression of SCN5A-E428K increased peak INa, but had no effect on late INa. (4) IKs and IKr interact, and hERG-E1039X caused a loss-of-function in IKs. (5) Immunocytochemical studies indicated that KCNQ1-R174C is trafficking defective and hERG-E1039X is defective in biosynthesis/degradation, but the abnormities were rescued by co-expression with WT. Thus, KCNQ1-R174C and hERG-E1039X disrupted IKs and IKr functions, respectively. The synergistic lesion, caused by KCNQ1-R174C and hERG-E1039X in IKs, is very likely why patients showed more severe phenotypes in the compound mutation case.

Phosphatidylinositol4-phosphate 5-kinase prevents the decrease in the HERG potassium current induced by Gq protein-coupled receptor stimulation.

The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG.

Ca2+/calmodulin potentiates I Ks in sinoatrial node cells by activating Ca2+/calmodulin-dependent protein kinase II.

The slow component of the delayed rectifier K(+) current (I Ks) plays an important role in the repolarization of action potentials in cardiac pacemaker cells and ventricular myocytes, and is regulated by various signaling pathways. Recent evidence has shown that calmodulin (CaM) is involved in modulation of diverse ion channels in cardiac myocytes under physiological and pathophysiological conditions. In the present study, we examined regulation of I Ks by Ca(2+)/CaM in guinea pig sinoatrial (SA) node cells using the whole-cell patch-clamp method. The density of I Ks was larger during intracellular dialysis with a higher Ca(2+) concentration (pCa 7, Ca (+)) compared to that with a low Ca(2+) concentration (pCa 10, Ca (-)). Intracellular application of CaM (400 nM) markedly potentiated I Ks with a Ca (+) pipette solution but not with a Ca (-) solution, thus showing that CaM potentiates I Ks in an intracellular Ca(2+)-dependent manner. Intracellular application of a specific Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, autocamtide-2 inhibitory peptide (AIP, 500 nM), markedly reduced I Ks activity in the presence of higher intracellular Ca(2+). Similarly, bath application of another inhibitor, KN-93 (1 µM) also significantly suppressed I Ks. Finally, the stimulatory action on I Ks of Ca(2+)/CaM was abolished by pretreatment with KN-93. Taken together, these observations suggest that Ca(2+)/CaM stimulates I Ks in guinea pig SA node cells through activation of CaMKII. This enhancement of I Ks by CaMKII may be involved in modulation of SA node automaticity under physiological or pathophysiological condition.

A novel HCN4 mutation, G1097W, is associated with atrioventricular block.

BACKGROUND: Loss-of-function mutations in the HCN4 gene have been shown to be associated with sinus dysfunction, but there are no reports on HCN4-mediated atrioventricular (AV) block. A novel missense HCN4 mutation G1097W was identified in a 69 year-old Japanese male with AV block, and we characterized the functional consequences of If-like channels reconstituted with the heterozygous HCN4 mutation. METHODS AND RESULTS: Wild-type (WT) HCN4 or/and HCN4-G1097W were expressed in a heterologous cell expression system. A functional assay using a whole-cell patch-clamp demonstrated that the mutant If-like currents were activated at more negative voltages compared to WT currents, while they retained the sensitivity to changes in intracellular cyclic adenosine monophosphate (cAMP) levels. Co-expression of G1097W with WT channels showed dominant-negative effects, including a reduction in peak currents and a negative voltage shifting on reconstituted currents. CONCLUSIONS: The HCN4-G1097W mutant channels displayed a loss-of-function type modulation on cardiac If channels and thus could predispose them to AV nodal dysfunction. These data provide a novel insight into the genetic basis for the AV block.

Gain-of-function KCNH2 mutations in patients with Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is an inherited disease characterized by right precordial ST segment elevation on electrocardiograms (ECGs) that predisposes patients to sudden cardiac death as a result of polymorphic ventricular tachyarrhythmia or ventricular fibrillation (VF). In BrS patients, except for SCN5A, mutations in other responsible genes are poorly elucidated. METHODS AND RESULTS: We identified 4 KCNH2 mutations, T152I, R164C, W927G, and R1135H, in 236 consecutive probands with BrS or Brugada-like ECG. Three of these mutation carriers showed QTc intervals shorter than 360 milliseconds and 1 experienced VF. We performed patch-clamp analyses on I(Kr) reconstituted with the KCNH2 mutations in Chinese hamster ovary cells and compared the phenotypes of the patients with different genotypes. Three mutations, R164C, W927G, and R1135H, increased I(Kr) densities. Three mutations, T152I, R164C, and W927G, caused a negative shift in voltage-dependent activation curves. Only the R1135H mutant channel prolonged the deactivation time constants. We also identified 20 SCN5A and 5 CACNA1C mutation carriers in our cohort. Comparison of probands' phenotypes with 3 different genotypes revealed that KCNH2 mutation carriers showed shorter QTc intervals and SCN5A mutation carriers had longer QRS durations. CONCLUSIONS: All KCNH2 mutations that we identified in probands with BrS exerted gain-of-function effects on I(Kr) channels, which may partially explain the ECG findings in our patients.

Cardiac channelopathies associated with infantile fatal ventricular arrhythmias: from the cradle to the bench.

BACKGROUND: Fatal ventricular arrhythmias in the early period of life have been associated with cardiac channelopathies for decades, and postmortem analyses in SIDS victims have provided evidence of this association. However, the prevalence and functional properties of cardiac ion channel mutations in infantile fatal arrhythmia cases are not clear. METHODS AND RESULTS: Seven infants with potentially lethal arrhythmias at age < 1 year (5 males, age of onset 44.1 ± 72.1 days) were genetically analyzed for KCNQ1, KCNH2, KCNE1-5, KCNJ2, SCN5A, GJA5, and CALM1 by using denaturing high-performance liquid chromatography and direct sequencing. Whole-cell currents of wildtype and mutant channels were recorded and analyzed in Chinese hamster ovary cells transfected with SCN5A and KCNH2 cDNA. In 5 of 7 patients, we identified 4 mutations (p.N1774D, p.T290fsX53, p.F1486del and p.N406K) in SCN5A, and 1 mutation (p.G628D) in KCNH2. N1774D, F1486del, and N406K in SCN5A displayed tetrodotoxin-sensitive persistent late Na(+) currents. By contrast, SCN5A-T290fsX53 was nonfunctional. KCNH2-G628D exhibited loss of channel function. CONCLUSION: Genetic screening of 7 patients was used to demonstrate the high prevalence of cardiac channelopathies. Functional assays revealed both gain and loss of channel function in SCN5A mutations, as well as loss of function associated with the KCNH2 mutation.

KCNE3 T4A as the genetic basis of Brugada-pattern electrocardiogram.

BACKGROUND: Brugada syndrome (BrS) is genetically heterogeneous. In Japanese BrS patients, except for SCN5A and KCNE5, mutations in the responsible genes have not yet been identified, and therefore the genetic heterogeneity remains poorly elucidated. METHODS AND RESULTS: Forty consecutive patients with Brugada-pattern electrocardiogram (ECG) underwent comprehensive genetic analysis of BrS-causing genes including SCN5A, SCN1B, SCN3B, CACNA1C, CACNB2, KCNE3 and KCNE5. Besides identifying 8 SCN5A mutations in the present cohort, a KCNE3 T4A mutation was found in a 55-year-old male patient who had experienced several episodes of syncope. A head-up tilt test during passive tilt provoked both hypotension and bradycardia, followed by syncope. He was therefore diagnosed with neurally mediated syncope (NMS). To characterize the functional consequence of the mutant, electrophysiological experiments using whole-cell patch-clamp methods and computer simulations using human right ventricular wall model were carried out. It was found that KCNE3 T4A increased I(to) recapitulated by heterologously coexpressing Kv4.3+KChIP2b+KCNE3-wild type or KCNE3-T4A in CHO cells. CONCLUSIONS: A KCNE3 T4A mutation was identified in a Japanese patient presenting Brugada-pattern ECG and NMS. Its functional consequence was the gain of function of I(to), which could underlie the pathogenesis of Brugada-pattern ECG. The data provide novel insights into the genetic basis of Japanese BrS.

GHGKHKNK octapeptide (P-5m) inhibits metastasis of HCCLM3 cell lines via regulation of MMP-2 expression in in vitro and in vivo studies.

P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3) in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC), and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2) activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.

Regulatory mechanisms underlying the modulation of GIRK1/GIRK4 heteromeric channels by P2Y receptors.

The muscarinic K(+) channel (I (K,ACh)) is a heterotetramer composed of GIRK1 (Kir3.1) and GIRK4 (Kir3.4) subunits of a G protein-coupled inwardly rectifying channel, and plays an important role in mediating electrical responses to the vagal stimulation in the heart. I (K,ACh) displays biphasic changes (activation followed by inhibition) through the stimulation of the purinergic P2Y receptors, but the regulatory mechanism involved in these modulation of I (K,ACh) by P2Y receptors remains to be fully elucidated. Various P2Y receptor subtypes and GIRK1/GIRK4 (I (GIRK)) were co-expressed in Chinese hamster ovary cells, and the effect of stimulation of P2Y receptor subtypes on I (GIRK) were examined using the whole-cell patch-clamp method. Extracellular application of 10 µM ATP induced a transient activation of I (GIRK) through the P2Y(1) receptor, which was completely abolished by pretreatment with pertussis toxin. ATP initially caused an additive transient increase in ACh-activated I (GIRK) (via M(2) receptor), which was followed by subsequent inhibition. This inhibition of I (GIRK) by ATP was attenuated by co-expression of regulator of G-protein signaling 2, or phosphatidylinositol-4-phosphate-5-kinase, or intracellular phosphatidylinositol 4,5-bisphosphate loading, but not by the exposure to protein kinase C inhibitors. P2Y(4) stimulation also persistently suppressed the ACh-activated I (GIRK). In addition, I (GIRK) evoked by the stimulation of the P2Y(4) receptor exhibited a transient activation, but that evoked by the stimulation of P2Y(2) or P2Y(12) receptor showed a rather persistent activation. These results reveal (1) that P2Y(1) and P2Y(4) are primarily coupled to the G(q)-phospholipase C-pathway, while being weakly linked to G(i/o), and (2) that P2Y(2) and P2Y(12) involve G(i/o) activation.

Atrioventricular block-induced Torsades de Pointes with clinical and molecular backgrounds similar to congenital long QT syndrome.

BACKGROUND: Atrioventricular block (AVB) sometimes complicates QT prolongation and torsades de pointes (TdP). METHODS AND RESULTS: The clinical and genetic background of 14 AVB patients (57±21 years, 13 females) who developed QT prolongation and TdP was analyzed. Electrophysiological characteristics of mutations were analyzed using heterologous expression in Chinese hamster ovary cells, together with computer simulation models. Every patient received a pacemaker or implantable cardioverter defibrillator; 3 patients had recurrence of TdP during follow-up because of pacing failure. Among the ECG parameters, QTc interval was prolonged to 561±76ms in the presence of AVB, but shortened to 495±42ms in the absence of AVB. Genetic screening for KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 revealed four heterozygous missense mutations of KCNQ1 or KCNH2 in 4 patients (28.6%). Functional analyses showed that all mutations had loss of functions and various gating dysfunctions of I(Ks) or I(Kr). Finally, action potential simulation based on the Luo-Rudy model demonstrated that most mutant channels induced bradycardia-related early afterdepolarizations. CONCLUSIONS: Incidental AVB, as a trigger of TdP, can manifest as clinical phenotypes of long QT syndrome (LQTS), and that some patients with AVB-induced TdP share a genetic background with those with congenital LQTS.

Presence and functional role of the rapidly activating delayed rectifier K(+) current in left and right atria of adult mice.

Repolarization of cardiac action potentials is regulated by several types of K(+) currents. The present study examined the presence and functional significance of rapid delayed rectifier (I(Kr)) in left and right atrial myocytes of mouse heart, using whole-cell patch-clamp method. The functional role of ultrarapid delayed rectifier (I(Kur)) in the repolarization was also examined by blocking with 4-aminopyridine (50 µM). The presence of I(Kr) was detected in left and right atrial myocytes as an E-4031 (5 µM)-sensitive current that exhibited relatively rapid activation during depolarization and half activation voltage of -17.5 and -17.4 mV for left and right atrial myocytes, respectively. The current density of I(Kr) was similar between left and right atria. The prolongation of action potential measured at 50% repolarization evoked by 4-aminopyridine was significantly larger in left than in right atrium, which appears to be consistent with the larger amplitude of I(Kur) in left atrium. On the other hand, the prolongation of action potential measured at 90% repolarization caused by E-4031 was significantly larger in right than in left atrium. The longer action potential of right atrium, which may result at least partly from smaller amplitude of I(Kur), is likely to enhance the functional significance of I(Kr) in repolarization process of right atrium, despite of similar magnitude of I(Kr) in left and right atria. Our data thus identifies I(Kr) in mouse atria and indicates the presence of functional interaction between I(Kr) and I(Kur) that potentially contributes to repolarization heterogeneity in left and right atria of mouse heart.

Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

Latent genetic backgrounds and molecular pathogenesis in drug-induced long-QT syndrome.

BACKGROUND: Drugs with I(Kr)-blocking action cause secondary long-QT syndrome. Several cases have been associated with mutations of genes coding cardiac ion channels, but their frequency among patients affected by drug-induced long-QT syndrome (dLQTS) and the resultant molecular effects remain unknown. METHODS AND RESULTS: Genetic testing was carried out for long-QT syndrome-related genes in 20 subjects with dLQTS and 176 subjects with congenital long-QT syndrome (cLQTS); electrophysiological characteristics of dLQTS-associated mutations were analyzed using a heterologous expression system with Chinese hamster ovary cells together with a computer simulation model. The positive mutation rate in dLQTS was similar to cLQTS (dLQTS versus cLQTS, 8 of 20 [40%] versus 91 of 176 [52%] subjects, P=0.32). The incidence of mutations was higher in patients with torsades de pointes induced by nonantiarrhythmic drugs than by antiarrhythmic drugs (antiarrhythmic versus others, 3 of 14 [21%] versus 5 of 6 [83%] subjects, P<0.05). When reconstituted in Chinese hamster ovary cells, KCNQ1 and KCNH2 mutant channels showed complex gating defects without dominant negative effects or a relatively mild decreased current density. Drug sensitivity for mutant channels was similar to that of the wild-type channel. With the Luo-Rudy simulation model of action potentials, action potential durations of most mutant channels were between those of wild-type and cLQTS. CONCLUSIONS: dLQTS had a similar positive mutation rate compared with cLQTS, whereas the functional changes of these mutations identified in dLQTS were mild. When I(Kr)-blocking agents produce excessive QT prolongation (dLQTS), the underlying genetic background of the dLQTS subject should also be taken into consideration, as would be the case with cLQTS; dLQTS can be regarded as a latent form of long-QT syndrome.

Adrenergic regulation of the rapid component of delayed rectifier K+ current: implications for arrhythmogenesis in LQT2 patients.

BACKGROUND: KCNH2 gene mutations disrupting rapid component of I(K) (I(Kr)) underlie type 2 congenital long QT syndrome (LQT2). Startled auditory stimuli are specific symptomatic triggers in LQT2, thus suggesting fast arrhythmogenic mechanism. OBJECTIVE: We investigated acute alpha(1A)- and cyclic adenosine monophosphate (cAMP)-related beta-adrenergic modulation of I(Kr) in HL-1 cardiomyocytes, wild type (WT)- and 2 LQT2-associated mutant Kv11.1 channels (Y43D- and K595E-Kv11.1) reconstituted in Chinese hamster ovary (CHO) cells. METHODS: I(Kr) and Kv11.1 currents were recorded using the whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with green fluorescent protein (GFP)-tagged pleckstrin homology domain of phospholipase C-delta(1) visualized fluctuations of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. RESULTS: In HL-1 cardiomyocytes expressing human alpha(1A)-adrenoceptor, superfusion with phenylephrine significantly reduced I(Kr) amplitude, shifted current activation to more positive potentials, and accelerated kinetics of deactivation. Confocal images showed a decline of membrane PIP(2) content during phenylephrine exposure. Simultaneous application of adenylyl cyclase activator forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) shifted I(Kr) activation to more negative potentials and decreased tail current amplitudes after depolarizations between +10 and +50 mV. In CHO cells, alpha(1A)-adrenoceptor activation downregulated WT-Kv11.1 channels and forskolin/IBMX produced a dual effect. Expressed alone, the Y43D-Kv11.1 or K595E-Kv11.1 channel had no measurable function. However, co-expression of WT-Kv11.1 and each mutant protein evoked currents with loss-of-function alterations but identical to WT-Kv11.1 alpha(1A)- and forskolin/IBMX-induced regulation. CONCLUSION: Acute adrenergic regulation of at least 2 Kv11.1 mutant channels is preserved as in WT-Kv11.1 and native I(Kr). Suppression of alpha(1A)-adrenoceptor-related transduction might have therapeutic implications in some cases of LQT2.

A novel KCNH2 mutation as a modifier for short QT interval.

In a 34-year-old man showing short QT interval (QTc 329 ms), we identified a novel C-terminal KCNH2 mutation, R1135H. Using a heterologous expression system with CHO cells, the mutant channels were found to display a significantly slow deactivation, which resulted in a gain-of-function for reconstituted 'I(Kr)' channels. This mutation could modify clinical phenotypes for this patient.

Hydroxyzine, a first generation H(1)-receptor antagonist, inhibits human ether-a-go-go-related gene (HERG) current and causes syncope in a patient with the HERG mutation.

QT prolongation, a risk factor for arrhythmias, can result from genetic variants in one (or more) of the genes governing cardiac repolarization as well as intake of drugs known to affect a cardiac K(+) channel encoded by human ether-a-go-go-related gene (HERG). In this paper, we will report a case of drug-induced long QT syndrome associated with an H(1)-receptor antagonist, hydroxyzine, in which a mutation was identified in the HERG gene. After taking 75 mg of hydroxyzine for several days, a 34-year-old female began to experience repetitive syncope. The deleterious effect of hydroxyzine was suspected because QTc interval shortened from 630 to 464 ms after cessation of the drug. Later on, the patient was found to harbor an A614V-HERG mutation. By using the patch-clamp technique in the heterologous expression system, we examined the functional outcome of the A614V mutation and confirmed a dominant-negative effect on HERG expression. Hydroxyzine concentration-dependently inhibited both wild-type (WT) and WT/A614V-HERG K(+) currents. Half-maximum block concentrations of WT and WT/A614V-HERG K(+) currents were 0.62 and 0.52 microM, respectively. Thus, accidental combination of genetic mutation and intake of hydroxyzine appeared to have led to a severe phenotype, probably, syncope due to torsade de pointes.

Regulation of the muscarinic K+ channel by extracellular ATP through membrane phosphatidylinositol 4,5-bisphosphate in guinea-pig atrial myocytes.

1 The present study was designed to examine the functional role of membrane phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the regulation of the muscarinic K(+) channel (I(K,ACh)) by extracellular ATP and adenosine in guinea-pig atrial myocytes, using the whole-cell patch-clamp method. 2 Bath application of ATP in micromolar concentrations typically evoked a transient activation of I(K,ACh); a rapid activation phase was consistently followed by a progressive decline even to the baseline level despite the continued presence of ATP. This progressive decline of I(K,ACh) was significantly attenuated either by blockade of phospholipase C (PLC) with compound 48/80 (100 microM) or by addition of PtdIns(4,5)P(2) (50 microM) to the cell inside, suggesting that depletion of membrane PtdIns(4,5)P(2) via PLC activation is mainly, if not totally, responsible for the progressive decline of I(K,ACh) during the presence of ATP. 3 When atrial myocytes were exposed to wortmannin (50 microM) following ATP (50 microM) application to impair the resynthesis of PtdIns(4,5)P(2), the activation of I(K,ACh) evoked by subsequently applied ATP (50 microM) was greatly reduced. Activation of I(K,ACh) by adenosine (100 microM) was partially reduced by pretreatment of atrial myocytes with ATP (100 microM) and was largely abolished by a further addition of wortmannin (50 microM) in the presence of ATP (100 microM). These results support the view that the activation of I(K,ACh) by ATP and adenosine depends on membrane PtdIns(4,5)P(2) that is subject to reduction by extracellular ATP. 4 The present study thus provides functional evidence to suggest that extracellular ATP activates PLC and thereby depletes membrane PtdIns(4,5)P(2) that is critically involved in the activation process of I(K,ACh) by its agonists ATP and adenosine in guinea-pig atrial myocytes.

Regulation of cardiac IKs potassium current by membrane phosphatidylinositol 4,5-bisphosphate.

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P2) was examined in guinea pig atrial myocytes using the whole-cell patch clamp method. IKs was elicited by depolarizing voltage steps given from a holding potential of -50 mV, and the effect of various test reagents on IKs was assessed by measuring the amplitude of tail current elicited upon return to the holding potential following a 2-s depolarization to +30 mV. Intracellular application of 50 microM wortmannin through a recording pipette evoked a progressive increase in IKs over a 10-15-min period to 208.5 +/- 14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane. Intracellular application of anti-PtdIns(4,5)P2 monoclonal antibody also increased the amplitude of IKs to 198.4 +/- 19.9% (n = 5). In contrast, intracellular loading with exogenous PtdIns(4,5)P2 at 10 and 100 mum produced a marked decrease in the amplitude of IKs to 54.3 +/- 3.8% (n = 5) and 44.8 +/- 8.2% (n = 5), respectively. Intracellular application of neomycin (50 microM) or aluminum (50 microM) evoked an increase in the amplitude of IKs to 161.0 +/- 13.5% (n = 4) and 150.0 +/- 8.2% (n = 4), respectively. These results strongly suggest that IKs channel is inhibited by endogenous membrane PtdIns(4,5)P2 through the electrostatic interaction with the negatively charged head group on PtdIns(4,5)P2. Potentiation of IKs by P2Y receptor stimulation with 50 microM ATP was almost totally abolished when PtdIns(4,5)P2 was included in the pipette solution, suggesting that depletion of membrane PtdIns(4,5)P2 is involved in the potentiation of IKs by P2Y receptor stimulation. Thus, membrane PtdIns(4,5)P2 may act as an important physiological regulator of IKs in guinea pig atrial myocytes.