Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome

IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel "flicker." At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was approximately 16 pS (corresponding to approximately 0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.     

Block by propofol and thiopentone of the min K current (IsK) expressed in Xenopus oocytes

The slowly activating component of the delayed rectifier potassium current (I(Ks)) in the heart is important during the repolarization of the cardiac action potential. Injection into Xenopus oocytes of mRNA coding for the min K protein induces a similar current (IsK) and recent observations support the hypothesis that functional channels result from the association of the min K protein with an endogenous K+ channel similar to the recently cloned KvLQT1. The general anaesthetics propofol and thiopentone have been shown to suppress cardiac I(Ks) with no effect on the rapidly activating component of I(K) (Takahashi and Terrar 1995). It was therefore of interest to test whether IsK was also inhibited by propofol and thiopentone. IsK was induced following injection into oocytes of min K mRNA which was transcribed in vitro from a synthetic gene (Hausdorff et al. 1991). IsK was activated by step depolarizations to a series of potentials from a holding potential of -40 mV and measured as the deactivating tail current on repolarization to the holding potential. Following a 2 s depolarization to +45 mV, propofol and thiopentone caused concentration-dependent reductions in IsK. The estimated IC50 value for the block of IsK by propofol was 250 microM and by thiopentone was 56 microM. Block of IsK by both propofol and thiopentone was not dependent on voltage or time. The reductions in IsK caused by propofol and thiopentone are consistent with the previously reported effects of these anaesthetics on I(Ks) in the heart and support the hypothesis that the min K protein contributes to the molecular basis of the cardiac I(Ks) channel.     

Activation and inactivation of homomeric KvLQT1 potassium channels

The voltage-gated potassium channel protein KvLQT1 (Wang et al., 1996. Nature Genet. 12:17-23) is believed to underlie the delayed rectifier potassium current of cardiac muscle together with the small membrane protein minK (also named IsK) as an essential auxiliary subunit (Barhanin et al., 1996. Nature. 384:78-80; Sanguinetti et al., 1996. Nature. 384:80-83) Using the Xenopus oocyte expression system, we analyzed in detail the gating characteristics of homomeric KvLQT1 channels and of heteromeric KvLQT1/minK channels using two-electrode voltage-clamp recordings. Activation of homomeric KvLQT1 at positive voltages is accompanied by an inactivation process that is revealed by a transient increase in conductance after membrane repolarization to negative values. We studied the recovery from inactivation and the deactivation of the channels during tail repolarizations at -120 mV after conditioning pulses of variable amplitude and duration. Most measurements were made in high extracellular potassium to increase the size of inward tail currents. However, experiments in normal low-potassium solutions showed that, in contrast to classical C-type inactivation, the inactivation of KvLQT1 is independent of extracellular potassium. At +40 mV inactivation develops with a delay of 100 ms. At the same potential, the activation estimated from the amplitude of the late exponential decay of the tail currents follows a less sigmoidal time course, with a late time constant of 300 ms. Inactivation of KvLQT1 is not complete, even at the most positive voltages. The delayed, voltage-dependent onset and the incompleteness of inactivation suggest a sequential gating scheme containing at least two open states and ending with an inactivating step that is voltage independent. In coexpression experiments of KvLQT1 with minK, inactivation seems to be largely absent, although biphasic tails are also observed that could be related to similar phenomena.     

Molecular basis of IsK protein regulation by oxidation or chelation

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.     

A model of cost-outcome analysis for assistive technology

Drosophila provides an excellent model for delineating the role of ion channels in the origin and transmission of heartbeat. We report here tests in Drosophila on a wide range of mutations and pharmacological agents known to interfere with K+, Ca2+, Na+, and Cl- ion channels in well-characterized ways. We find K+ channels are central to heart function. Tetraethylammonium, which blocks all four K+ currents, slowed the heart. We were able to distinguish among these currents. The mutation slowpoke and the agent charybdotoxin, both of which affect a fast Ca(2+)-gated K+ channel, virtually eliminate heartbeat. Shaker and ether-a-go-go, which encode subunits of K+ channels, have moderate, possibly regulatory effects. "OPQ-type" Ca2+ channels are critical. omega-Conotoxin MVIIC, which blocks these channels, virtually stops the heart. Amiloride, which may affect T-type Ca2+ channels, has no effect, nor do the L-type Ca2+ blockers verapamil and diltiazem. temperature induced paralysis E, involved in the function of Na+ channels, the Na+ channel blockers tetrodotoxin and amiloride, and the Cl- blockers mefanamic and niflumic acids have no effect. Na+ and Cl- channels thus appear unnecessary for cardiac function.     

Tissue selectivity of calcium channel blockers

Calcium channel blockers are also termed calcium antagonists or calcium entry blockers. The use of calcium antagonists for the management of hypertension is well established. Their control of vascular tone is related to their interaction with the alpha 1 subunit of L-type calcium channels. This interaction is not simple since prolonged depolarisation promotes the inactivated state of the channels resulting in a change of affinity which is different for various molecules so far considered. The isoforms of alpha 1 subunits and the duration of the stimulus required to activate heart or vessels are important parameters to be considered with the nature of the molecule. Those parameters influence the vascular selectivity which is quantified as the ratio of the concentrations required to reduce by 50% the contraction of heart and of vessels. This selectivity is an important component in the therapeutic action. Another component of this action is the prevention of structural changes noted in heart and arteries. As well as lowering blood pressure, calcium channel blockers have also been found to exert blood pressure independent effects. For instance, they reduce cardiac and vascular hypertrophy and avoid renal damage. In the stroke-prone rat, such protective effects are accompanied by reduction of the salt-dependent overexpression of the gene of endothelin-1 and of fetal genes associated with cardiac hypertrophy. This paper summarizes available information about those components and discuss their significance.     

Dominant-negative KvLQT1 mutations underlie the LQT1 form of long QT syndrome

BACKGROUND: Mutations that map to the KvLQT1 gene on human chromosome 11 account for more than 50% of inherited long QT syndrome (LQTS). It has been discovered recently that the KvLQT1 and minK proteins functionally interact to generate a current with biophysical properties similar to I(Ks), the slowly activating delayed-rectifier cardiac potassium current. Since I(Ks) modulates the repolarization of cardiac action potentials it is reasonable to hypothesize that mutations in KvLQT1 reduce I(Ks), resulting in the prolongation of cardiac action potential duration. METHODS AND RESULTS: We expressed LQTS-associated KvLQT1 mutants in Xenopus oocytes either individually or in combination with wild-type KvLQT1 or in combination with both wild-type KvLQT1 and minK. Substitutions of alanine with proline in the S2-S3 cytoplasmic loop (A177P) or threonine with isoleucine in the highly conserved signature sequence of the pore (T311I) yield inactive channels when expressed individually, whereas substitution of leucine with phenylalanine in the S5 transmembrane domain (L272F) yields a functional channel with reduced macroscopic conductance. However, all these mutants inhibit wild-type KvLQT1 currents in a dominant-negative fashion. CONCLUSIONS: In LQTS-affected individuals these mutations would be predicted to result in a diminution of the cardiac I(Ks) current, subsequent prolongation of cardiac repolarization, and an increased risk of arrhythmias.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.     

Functional expression of two KvLQT1-related potassium channels responsible for an inherited idiopathic epilepsy

Benign familial neonatal convulsions (BFNC), a class of idiopathic generalized epilepsy, is an autosomal dominantly inherited disorder of newborns. BFNC has been linked to mutations in two putative K+ channel genes, KCNQ2 and KCNQ3. Amino acid sequence comparison reveals that both genes share strong homology to KvLQT1, the potassium channel encoded by KCNQ1, which is responsible for over 50% of inherited long QT syndrome. Here we describe the cloning, functional expression, and characterization of K+ channels encoded by KCNQ2 and KCNQ3 cDNAs. Individually, expression of KCNQ2 or KCNQ3 in Xenopus oocytes elicits voltage-gated, rapidly activating K+-selective currents similar to KCNQ1. However, unlike KCNQ1, KCNQ2 and KCNQ3 currents are not augmented by coexpression with the KCNQ1 beta subunit, KCNE1 (minK, IsK). Northern blot analyses reveal that KCNQ2 and KCNQ3 exhibit similar expression patterns in different regions within the brain. Interestingly, coexpression of KCNQ2 and KCNQ3 results in a substantial synergistic increase in current amplitude. Coexpression of KCNE1 with the two channels strongly suppressed current amplitude and slowed kinetics of activation. The pharmacological and biophysical properties of the K+ currents observed in the coinjected oocytes differ somewhat from those observed after injecting either KCNQ2 or KCNQ3 by itself. The functional interaction between KCNQ2 and KCNQ3 provides a framework for understanding how mutations in either channel can cause a form of idiopathic generalized epilepsy.     

The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C

A clone encoding the guinea pig (gp) min K potassium channel was isolated and expressed in Xenopus oocytes. The currents, gpIsK, exhibit many of the electrophysiological and pharmacological properties characteristic of gpIKs, the slow component of the delayed rectifier potassium conductance in guinea pig cardiac myocytes. Depolarizing commands evoke outward potassium currents that activate slowly, with time constants on the order of seconds. The currents are blocked by the class III antiarrhythmic compound clofilium but not by the sotalol derivative E4031 or low concentrations of lanthanum. Like IKs in guinea pig myocytes, gpIsK is modulated by stimulation of protein kinase A and protein kinase C (PKC). In contrast to rat and mouse IsK, which are decreased upon stimulation of PKC, myocyte IK and gpIsK in oocytes are increased after PKC stimulation. Substitution of an asparagine residue at position 102 by serine (N102S), the residue found in the analogous position of the mouse and rat min K proteins, results in decreased gpIsK in response to PKC stimulation. These results support the hypothesis that the min K protein underlies the slow component of the delayed rectifier potassium current in ventricular myocytes and account for the species-specific responses to stimulation of PKC.     

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.     

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.     

Structures and functions of calcium channel beta subunits

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.     

Differential effect of beta-adrenergic stimulation on the frequency-dependent electrophysiologic actions of the new class III antiarrhythmics dofetilide, ambasilide, and chromanol 293B

INTRODUCTION: Blockade of the rapid delayed rectifier potassium current (IKr) as an important mechanism for current Class III antiarrhythmics is less effective in action potential prolongation during beta-adrenergic activation. We hypothesized that blockade of the increased slow IK (IKs) current during beta-adrenergic stimulation could improve action potential prolongation and tested this hypothesis by comparison of three different IK blockers: dofetilide, a selective blocker of IKr; ambasilide, a nonselective blocker of IK; and chromanol 293B, a selective blocker of IKs. METHODS AND RESULTS: Transmembrane action potential duration was determined in guinea pig papillary muscles. After equilibration with the potassium channel blockers (dofetilide 10 nM, ambasilide 10 microM, chromanol 293B 10 microM), isoproterenol (10 and 100 nM) was added. The action potential prolonging effect of dofetilide was reduced in the presence of increasing concentrations of isoproterenol whereas the effect of ambasilide was much less reduced. In contrast, the effect of chromanol 293B clearly was increased in the presence of both concentrations of isoproterenol. No afterdepolarizations were observed after application of isoproterenol in control. Following isoproterenol, but not before, dofetilide and chromanol 293B induced early afterdepolarizations in 20% and 17% of the papillary muscles, whereas ambasilide and chromanol 293B induced delayed afterdepolarizations in 27% and 33%, respectively. CONCLUSION: In contrast to dofetilide, the Class III effect of ambasilide is less reduced and the effect of chromanol 293B is enhanced during beta-adrenergic stimulation. Our data support the hypothesis that IKs blockade improves the efficacy of antiarrhythmics in action potential prolongation during beta-adrenergic activation; however, this effect may increase the risk of afterdepolarizations.     

A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids

TWIK-1, TREK-1 and TASK K+ channels comprise a class of pore-forming subunits with four membrane-spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK-1, TREK-1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non-inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid-stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.