HERG-like K+ channels in microglia

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.     

Idiosyncratic gating of HERG-like K+ channels in microglia

A simple kinetic model is presented to explain the gating of a HERG-like voltage-gated K+ conductance described in the accompanying paper (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:781-794). The model proposes two kinetically distinct closing pathways, a rapid one favored by depolarization (deactivation) and a slow one favored by hyperpolarization (inactivation). The overlap of these two processes leads to a window current between -50 and +20 mV with a peak at -36 mV of approximately 12% maximal conductance. The near absence of depolarization-activated outward current in microglia, compared with HERG channels expressed in oocytes or cardiac myocytes, can be explained if activation is shifted negatively in microglia. As seen with experimental data, availability predicted by the model was more steeply voltage dependent, and the midpoint more positive when determined by making the holding potential progressively more positive at intervals of 20 s (starting at -120 mV), rather than progressively more negative (starting at 40 mV). In the model, this hysteresis was generated by postulating slow and ultra-slow components of inactivation. The ultra-slow component takes minutes to equilibrate at -40 mV but is steeply voltage dependent, leading to protocol-dependent modulation of the HERG-like current. The data suggest that "deactivation" and "inactivation" are coupled through the open state. This is particularly evident in isotonic Cs+, where a delayed and transient outward current develops on depolarization with a decay time constant more voltage dependent and slower than the deactivation process observed at the same potential after a brief hyperpolarization.     

Blockade of the human cardiac K+ channel Kv1.5 by the antibiotic erythromycin

The Internet, as a global computer network, provides opportunities to make available multimedia educational materials, such as teaching files and image databases, that can be accessed using "World-Wide Web" client browser to provide continuing medical education. Since August, 1995, at the Institute of Radiology-University of Palermo, we developed a World-Wide Web server on the Internet to provide a collection of interactive radiology educational resources such as teaching files and image database for continuing medical education in radiology. Our server is based on a UNIX workstation connected to the Internet via our campus Ethernet network and reachable at the uniform resource locator (URL) address: http:/(/)mbox.unipa.it/approximately radpa/ radpa.html. Digital CT and MR images for teaching files and image database are downloaded through an Ethernet local area network from a GE Advantage Windows workstation. US images will be acquired on-line through a video digitizing board. Radiographs will be digitized by means of a Charge Coupled Device (CCD) scanner. To set up teaching files, image database and all other documents, we use the standard "HyperText Markup Language" (HTML) to edit the documents, and the Graphics Interchange Format (GIF) or Joint Photographic Expert Group (JPEG) format to store the images. Nine teaching files are presently available on the server, together with 49 images in the database, a list of international radiological servers, a section devoted to the museum of radiology hosted by our Institute, the electronic version of the Journal Eido Electa. In the first 12 months of public access through the Internet, 12,280 users accessed the server worldwide: 45% of them to retrieve teaching files; 35% to retrieve images from the database; the remaining 20% to retrieve other documents. Placing teaching files and image database on a World-Wide Web server makes these cases more available to residents and radiologists to provide continuing medical education in radiology.     

Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation

Chronic atrial fibrillation is associated with a shortening of the atrial action potential duration and atrial refractory period. To test the hypothesis that these changes are mediated by changes in the density of specific atrial K+ currents, we compared the density of K+ currents in left and right atrial myocytes and the density of delayed rectifier K+ channel alpha-subunit proteins (Kv1.5 and Kv2.1) in left and right atrial appendages from patients (n = 28) in normal sinus rhythm with those from patients (n = 15) in chronic atrial fibrillation (AF). Contrary to our expectations, nystatin-perforated patch recordings of whole-cell K+ currents revealed significant reductions in both the inactivating (ITO) and sustained (IKsus) outward K+ current densities in left and right atrial myocytes isolated from patients in chronic AF, relative to the ITO and IKsus densities in myocytes isolated from patients in normal sinus rhythm. Quantitative Western blot analysis revealed that although there was no change in the expression of the Kv2.1 protein, the expression of Kv1.5 protein was reduced by > 50% in both the left and the right atrial appendages of AF patients. The finding that Kv1.5 expression is reduced in parallel with the reduction in delayed rectifier K+ current density is consistent with recent suggestions that Kv1.5 underlies the major component of the delayed rectifier K+ current in human atrial myocytes, the ultrarapid delayed rectifier K+ current, IKur. The unexpected finding of reduced voltage-gated outward K+ current densities in atrial myocytes from AF patients demonstrates the need to further examine the details of the electrophysiological remodeling that occurs during AF to enable more effective and safer therapeutic strategies to be developed.     

Expression environment determines K+ current properties: Kv1 and Kv4 alpha-subunit-induced K+ currents in mammalian cell lines and cardiac myocytes

Voltage-gated K+ channel (Kv) pore-forming (alpha) subunits of the Kv1 and Kv4 subfamilies have been cloned from heart cDNA libraries, and are thought to play roles in the generation of the transient outward K+ current, Ito. Heterologous expression of these subunits in Xenopus oocytes, however, reveals K+ currents that are quite distinct from Ito. In the experiments here, the detailed time- and voltage-dependent properties of the currents expressed in mammalian cell lines and in cardiac myocytes by Kv1.4 and Kv4.2 were examined and compared to previous findings in studies of oocytes, as well as to Ito characterized in various myocardial cells. As in oocytes, expression of Kv1.4 in HEK-293, Ltk- or neonatal rat ventricular cells reveals rapidly activating K+ currents. In contrast to the currents in oocytes, however, there are two components of inactivation of the Kv1.4-induced currents in mammalian cells, and both components are significantly slower in myocytes than in either HEK-293 or Ltk- cells. In addition, in all three cell types, recovery of Kv1.4 from steady-state inactivation is very slow, proceeding with mean time constants in the range of 6-8 s. The properties of Kv4.2-induced currents also vary with cell type and, importantly, the rates of activation, inactivation and recovery from inactivation are significantly faster in mammalian cells than in Xenopus oocytes. In HEK-293, Chinese hamster ovary (CHO) and neonatal rat ventricular cells, for example, the currents recover from steady-state inactivation with mean (+/-SD) time constants of 153+/-32 (n=12), 245+/-112 (n=10) and 86+/-38 (n=11) ms, respectively; therefore, recovery proceeds 5-10 times faster than observed for Kv4.2 in oocytes. These results emphasize the importance of the cellular expression environment in efforts to correlate endogenous K+ currents with heterologously expressed K+ channel subunits. In addition, the finding that Kv alpha subunits produce distinct K+ currents in different cells suggests that cell-type-specific associations with endogenous Kv alpha or accessory beta subunits and/or post-translational processing play roles in determining the properties of functional K+ channels.     

Functional effects of expression of hslo Ca2+ activated K+ channels in cultured macrovascular endothelial cells

OBJECTIVE: The measurement of asthma, rhinitis and eczema have been subject of controversy due to lack of a standardized methodology. To test the applicability of a standardized methodology for comparisons of time and space we determined the prevalence of asthma and other allergic diseases in a random sample of schoolchildren (n = 6,238) from 6 to 8 and 11 to 14 years of age living in Cuernavaca, Morelos, Mexico. MATERIAL AND METHODS: The methodology proposed by the International Study of Asthma and Allergies in Childhood (ISAAC) to determine prevalence and severity of asthma, rhinitis and eczema was applied. Current and accumulated information on prevalence was obtained by means of a standardized questionnaire answered by the children's parents. RESULTS: The accumulated prevalence of asthma by medical diagnosis and wheezing was 5.8% (5.2-6.4) and 21.8% (20.7-22.9) respectively; prevalence of wheezing in the last 12 months was 8.9% in the group of 6 to 8 years against 6.6% in the 11 to 14 year old group p < 0.001. Prevalence of the medical diagnosis of rhinitis was 4.9% (4.3-5.5). Regarding the typical symptoms of rhinitis, in the last 12 months prevalence was 9.6% (6-8 years) and 10.1% (11-14 years). Prevalence of eczema by medical diagnosis was 4.1% (3.6-4.6). Prevalence of eczema symptoms in the last 12 months was 10.1% (6-8 years) and 10.6% (11-14 years). Prevalence of severe asthma symptoms was significantly higher in the 6 to 8 year olds and in the autumn. CONCLUSIONS: Prevalence of asthma by medical diagnosis and by symptoms is relatively low with respect to other studies performed with the same methodology. The benefits of using a standardized methodology were analyzed.     

Developmental regulation of neuronal K+ channels by target-derived TGF beta in vivo and in vitro

The functional expression of Ca2+-activated K+ channels (KCa) in developing chick ciliary ganglion (CG) neurons requires interactions with target tissues and preganglionic innervation. Here, we show that the stimulatory effects of target tissues are mediated by an isoform of TGFbeta. Exposure of cultured CG neurons to TGFbeta1, but not TGFbeta2 or TGFbeta3, caused robust stimulation of KCa. The KCa stimulatory effects of target tissue extracts were blocked by a neutralizing pan-TGFbeta antiserum but not by specific TGFbeta2 or TGFbeta3 antisera. Intraocular injection of TGFbeta1 caused robust stimulation of KCa, whereas intraocular injection of pan-TGFbeta antiserum inhibited expression of KCa in CG neurons developing in vivo. The effects of TGFbeta1 were potentiated by beta-neuregulin-1, a differentiation factor expressed in preganglionic neurons.     

Inactivation of voltage-gated cardiac K+ channels

Inactivation is the process by which an open channel enters a stable nonconducting conformation after a depolarizing change in membrane potential. Inactivation is a widespread property of many different types of voltage-gated ion channels. Recent advances in the molecular biology of K+ channels have elucidated two mechanistically distinct types of inactivation, N-type and C-type. N-type inactivation involves occlusion of the intracellular mouth of the pore through binding of a short segment of residues at the extreme N-terminal. In contrast to this "tethered ball" mechanism of N-type inactivation, C-type inactivation involves movement of conserved core domain residues that result in closure of the external mouth of the pore. Although C-type inactivation can show rapid kinetics that approach those observed for N-type inactivation, it is often thought of as a slowly developing and slowly recovering process. Current models of C-type inactivation also suggest that this process involves a relatively localized change in conformation of residues near the external mouth of the permeation pathway. The rate of C-type inactivation and recovery can be strongly influenced by other factors, such as N-type inactivation, drug binding, and changes in [K+]o. These interactions make C-type inactivation an important biophysical process in determining such physiologically important properties as refractoriness and drug binding. C-type inactivation is currently viewed as arising from small-scale rearrangements at the external mouth of the pore. This review will examine the multiplicity of interactions of C-type inactivation with N-terminal-mediated inactivation and drug binding that suggest that our current view of C-type inactivation is incomplete. This review will suggest that C-type inactivation must involve larger-scale movements of transmembrane-spanning domains and that such movements contribute to the diversity of kinetic properties observed for C-type inactivation.     

Inhibition of cAMP mediated relaxation in rat coronary vessels by block of Ca++ activated K+ channels

The hypothesis for this study is that block of calcium activated potassium (KCa) channels inhibits cAMP induced relaxation in pressurized rat coronary resistance arteries. Pressure-diameter experiments with septal arteries (200-270 microns internal diameter at 60 mmHg and maximum dilation) showed significant basal tone over a range of pressure from 40-120 mmHg. The level of tone was increased with the thromboxane A2 analogue 9,11-dideoxy-11 alpha, 9 alpha-epoxy-methanoprostaglandin F2 alpha (U46619) in all experiments. Receptor activation of the cAMP pathway was done with adenosine (ADO) and isoproterenol (ISO). Tetraethylammonium ion (TEA+), 1mM, significantly inhibited relaxation to ADO (10(-6)-10(-3)M) with a maximal inhibition of 75 +/- 7% (as a % of maximum diameter change with the vasodilator alone) at 10(-3)M ADO. TEA+ inhibited ISO (10(-6)M) relaxation by 63 +/- 9%. Direct activation of the cAMP pathway was done with forskolin and 8-bromo-cAMP. TEA+ significantly inhibited forskolin (10(-6)-10(-4)M) induced relaxation with a maximal inhibition of 81.3 +/- 1.2% at 10(-4)M forskolin. TEA+ and iberiotoxin (10(-7)M) significantly inhibited 8- bromo-cAMP (10(-3)M) induced relaxation by 72 +/- 5% and 56 +/- 3% respectively. The effect of TEA+ on relaxation induced by nitroprusside (a cGMP dependent vasodilator) was not significant. The results show that rat coronary resistance arteries possess significant myogenic tone and modulation of Kca channels plays a major role in cAMP mediated relaxation.     

Inhibition of cGMP mediated relaxation in small rat coronary arteries by block of CA++ activated K+ channels

Children with sleep disorders are often inattentive or hyperactive, and some carry a diagnosis of attention deficit/hyperactivity disorder (ADHD) until their sleep disorder is detected. However, the potential behavioral impact of undiagnosed sleep disorders is not known. We sought to determine whether children with higher levels of inattention and hyperactivity more frequently have symptoms of sleep-related breathing disorders (SRBDs) or periodic limb movement disorder (PLMD). We surveyed parents of 2-18-year-old patients at a child psychiatry clinic (n = 70) and a general pediatrics clinic (n = 73) to assess the children's behavior, snoring, complaints of restless legs at night, and daytime sleepiness. A validated pediatric sleep questionnaire provided the explanatory variables, and a scale for inattention and hyperactivity, derived from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV), provided the dependent variable. Habitual snoring was more frequent (33%) among children who carried a diagnosis of ADHD than among the other children at the psychiatry or general pediatric clinics (11 and 9%, respectively, chi-square test, p = 0.01). Snoring scores, derived from six snoring- and SRBD-related question items, were associated with higher levels of inattention and hyperactivity. The complaint of restless legs and a composite score for daytime sleepiness showed some evidence, though less consistent, of an association with inattention and hyperactivity. The association of snoring with inattention and hyperactivity suggests that SRBDs and perhaps other sleep disorders could be a cause of inattention and hyperactivity in some children. If a causal effect is present, our data suggest that 81% of habitually snoring children who have ADHD--25% of all children with ADHD--could have their ADHD eliminated if their habitual snoring and any associated SRBD were effectively treated.     

Blockade of HERG and Kv1.5 by ketoconazole

Ketoconazole, a widely used fungicide in patients, has been associated with Q-T prolongation and torsade de pointes when co-administered with terfenadine (Seldane). Both compounds use the same cytochrome-P450 metabolic pathway, resulting in an increase in plasma concentration of terfenadine. We previously showed that terfenadine blocked HERG (Human Ether-a-Gogo Related Gene), an important component of the repolarizing cardiac delayed rectifier IK with concentration needed to obtain 50% of the block (IC50) in the therapeutic range (300 nM). Another target is Kv1.5 (delayed outward rectifier potassium current), an important component of human atrial ultrarapid delayed rectifier current. Whether Kv1.5 and HERG proteins are direct targets for ketoconazole has yet to be addressed. We heterologously expressed HERG and Kv1.5 in Xenopus oocytes and compared their sensitivities to ketoconazole. HERG and Kv1.5 currents were reduced comparably with apparent IC50 values of 49 microM and 107 microM, respectively, when measured using the two-microelectrode recording technique. The differences in the IC50 may help explain the preferential ventricular origin of the ketoconazole-associated arrhythmias during overdose. The mechanism of block was different between Kv1.5 and HERG. Cumulative application of terfenadine and ketoconazole at their respective IC50 concentrations resulted in current reductions that suggest an additive rather than a competitive type of block by the two drugs. We conclude that ketoconazole may potentiate the effects of terfenadine first by an indirect pharmacokinetic action to elevate plasma levels and second by a direct pharmacodynamic action on HERG currents. These potential dual actions on HERG currents suggest that precautions should be taken in long-term ketoconazole treatment, particularly for patients who have decreased liver function or are on a drug regimen requiring simultaneous medications that use cytochrome-P450 for breakdown, such as terfenadine or erythromycin, or Class III antiarrhythmic drugs.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Decreased expression of Kv4.2 and novel Kv4.3 K+ channel subunit mRNAs in ventricles of renovascular hypertensive rats

Hypertension-induced cardiac hypertrophy is associated with alterations in ventricular action potentials. To understand molecular mechanisms underlying this electrical abnormality, expression of cardiac voltage-gated K+ channel subunit genes was examined in ventricles of renovascular hypertensive rats. While generating a rat Kv4.3 probe, we discovered a previously unreported 19-amino acid insertion in the C-terminal intracellular region of the channel subunit. RNase protection assays indicated that this novel isoform is predominant in rat lung and heart. Effects of renovascular hypertension were then determined by using renal artery clipping models: two-kidney, one clip (2K-1C) rats, a model of high-renin hypertension with a normal plasma volume, and one-kidney, one clip (1K-1C) rats, a model of normal renin with a raised plasma volume. Expression of Kv4.2 and Kv4.3 mRNAs was diminished by > 50% in ventricles of 2K-1C rats; however, no changes in the expression of Kv1.2, Kv1.4, Kv1.5, Kv2.1, or KvLQT1 mRNAs were detected. Similar downregulation of Kv4.2 and Kv4.3 mRNAs was detected in 1K-1C rats. Chronic administration of captopril, an angiotensin-converting enzyme inhibitor, blocked the development of hypertension and the suppression of Kv4 subfamily channel mRNA expression in 2K-1C rats. Furthermore, captopril administration to sham-operated rats significantly increased Kv4.2 mRNA. These results indicate that renovascular hypertension causes specific reductions in Kv4 subfamily channel mRNA expression and that this effect is likely to be mediated primarily by an increase in cardiac afterload.     

Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels

ATP-sensitive K+ (K(ATP)) channels are nucleotide-gated channels that couple the metabolic status of a cell with membrane excitability and regulate a number of cellular functions, including hormone secretion and cardioprotection. Although intracellular ATP is the endogenous inhibitor of K(ATP) channels and ADP serves as the channel activator, it is still a matter of debate whether changes in the intracellular concentrations of ATP, ADP, and/or in the ATP/ADP ratio could account for the transition from the ATP-liganded to the ADP-liganded channel state. Here, we overview evidence for the role of cellular phosphotransfer cascades in the regulation of K(ATP) channels. The microenvironment of the K(ATP) channel harbors several phosphotransfer enzymes, including adenylate, creatine, and pyruvate kinases, as well as other glycolytic enzymes that are able to transfer phosphoryls between ATP and ADP in the absence of major changes in cytosolic levels of adenine nucleotides. These phosphotransfer reactions are governed by the metabolic status of a cell, and their phosphotransfer rate closely correlates with K(ATP) channel activity. Adenylate kinase catalysis accelerates the transition from ATP to ADP, leading to K(ATP) channel opening, while phosphotransfers driven by creatine and pyruvate kinases promote ADP to ATP transition and channel closure. Thus, through delivery and removal of adenine nucleotides at the channel site, phosphotransfer reactions could regulate ATP/ADP balance in the immediate vicinity of the channel and thereby the probability of K(ATP) channel opening. In this way, phosphotransfer reactions could provide a transduction mechanism coupling cellular metabolic signals with K(ATP) channel-associated functions.     

Signalling via the G protein-activated K+ channels

The inwardly rectifying K+ channels of the GIRK (Kir3) family, members of the superfamily of inwardly rectifying K+ channels (Kir), are important physiological tools to regulate excitability in heart and brain by neurotransmitters, and the only ion channels conclusively shown to be activated by a direct interaction with heterotrimeric G protein subunits. During the last decade, especially since their cloning in 1993, remarkable progress has been made in understanding the structure, mechanisms of gating, activation by G proteins, and modulation of these channels. However, much of the molecular details of structure and of gating by G protein subunits and other factors, mechanisms of modulation and desensitization, and determinants of specificity of coupling to G proteins, remain unknown. This review summarizes both the recent advances and the unresolved questions now on the agenda in GIRK studies.     

Comparative effects of nonsedating histamine H1 receptor antagonists, ebastine and terfenadine, on human Kv1.5 channels

The effects of ebastine and terfenadine, long-acting nonsedating histamine H1 receptor antagonists, were studied on hKv1.5 channels using the whole-cell voltage-clamp configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding the hKv1.5 channel. Upon depolarization to +60 mV, terfenadine, 1 microM and 3 microM, inhibited the hKv1.5 current by 42.4 +/- 6.4% and 69.3 +/- 4.2% (P < 0.01). In contrast, at the same range of concentrations, ebastine-induced inhibition of this K+ current averaged 6.5 +/- 2.0% and 13.0 +/- 2.0 (P < 0.05). At the highest concentration tested (3 microM) neither terfenadine carboxylate nor carebastine significantly modified hKv1.5 current. All these results suggest that ebastine could represent a safer alternative to terfenadine in the clinical practice.     

Regulation of Kv4.2 and Kv1.4 K+ channel expression by myocardial hypertrophic factors in cultured newborn rat ventricular cells

Postnatal development and myocardial hypertrophy are associated with alterations in cardiac voltage-gated K+ channels. To investigate mechanisms underlying this K+ channel remodeling, expression of Kv4.2 and Kv1.4 K+ channel alpha-subunits was examined in cultured newborn rat ventricular myocytes by Western blot analysis using polyclonal antibodies against each of the subunits. At day 5 of cell culture, Kv1.4 protein was expressed at higher level than Kv4.2; as the age of culture progressed, Kv1.4 was significantly diminished while Kv4.2 increased with time in culture and became the predominant K+ channel protein. Such K+ channel isoform switch from Kv1.4 to Kv4.2 resembles that of the development in vivo. A 72-h treatment with exogenous triiodothyronine (T3, 0.1 microM) to cultured neonatal myocytes enhanced the expression of Kv4.2 by 73% and decreased the Kv1.4 expression by 22%. The effects of T3 were associated with an increase in the protein-to-DNA ratio indicating myocyte hypertrophy. On the other hand, a 72-h treatment with cardiac non-myocyte cell (NMC)-conditioned growth medium (NCGM) or phenylephrine (20 microM) induced similar cell hypertrophy, but in sharp contrast to T3, both markedly suppressed the Kv4.2 channel protein level. In addition, the trophic and the Kv4.2-downregulating effects of NCGM could be mimicked by exogenous endothelin-1 (0.1 microM), a paracrine factor secreted from cardiac NMCs. Our observations for the first time suggest that cardiac Kv4.2 and Kv1.4 K+ channel alpha-subunits are differentially regulated by a variety of myocardial hypertrophic factors. That T3 accelerated the developmental K+ channel isoform switch from Kv1.4 to Kv4.2 in vitro indicates the critical importance of thyroid hormone in postnatal K+ channel remodeling. Cardiac NMCs and alpha-adrenoceptor activation may contribute to the reduced outward K+ channel density in hypertrophied cardiomyocytes.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Glibenclamide-sensitive K+ channels underlying levcromakalim-induced relaxation in pig urethra

To investigate the possible mechanisms involved in the stable and long-lasting levcromakalim-induced relaxation of the resting urethral tone, we have performed mechanical and voltage-clamp experiments using intact tissue and isolated cells from pig urethra, respectively. At negative membrane potentials, levcromakalim induced time- and voltage-independent membrane currents in whole-cell configurations. In cell-attached patches, levcromakalim not only increased the open-state probability (the NP(0) value) of the glibenclamide-sensitive 43 pS K+ channel (K(GS)) in a concentration-dependent manner, but also activated K(GS) with a time- and voltage-independence. During long burst-like channel activity, neither the mean open lifetime nor the mean closed time of K(GS) exhibited voltage-dependency between -100 and - 40 mV. It is concluded that levcromakalim causes a stable and potent relaxation of pig urethra through opening of K(GS) which possesses time- and voltage-independent activating mechanisms.