Mutations in the hminK gene cause long QT syndrome and suppress IKs function.

Ion-channel beta-subunits are ancillary proteins that co-assemble with alpha-subunits to modulate the gating kinetics and enhance stability of multimeric channel complexes. Despite their functional importance, dysfunction of potassium-channel beta-subunits has not been associated with disease. Recent physiological studies suggest that KCNE1 encodes beta-subunits (hminK) that co-assemble with KvLQT1 alpha-subunits to form the slowly activating delayed rectifier K+ (IKs) channel. Because KVLQT1 mutations cause arrhythmia susceptibility in the long QT syndrome (LQT), we hypothesized that mutations in KCNE1 also cause this disorder. Here, we define KCNE1 missense mutations in affected members of two LQT families. Both mutations (S74L, D76N) reduced IKs by shifting the voltage dependence of activation and accelerating channel deactivation. D76N hminK also had a strong dominant-negative effect. The functional consequences of these mutations would be delayed cardiac repolarization and an increased risk of arrhythmia. This is the first description of KCNE1 as an LQT gene and confirms that hminK is an integral protein of the IKs channel.

Molecular genetic insights into cardiovascular disease.

The recent application of molecular genetic tools to inherited forms of cardiovascular disease has provided important insight into the molecular mechanisms underlying cardiac arrhythmias, cardiomyopathies, and vascular diseases. These studies point to defects in ion channels, contractile proteins, structural proteins, and signaling molecules as key players in disease pathogenesis. Genetic testing is now available for a subset of inherited cardiovascular diseases, and new mechanism-based therapies may be available in the near future. This remarkable progress and the implications it may have for more common forms of cardiovascular disease are reviewed here.

Pathophysiology of ion channel mutations.

The past year has seen significant advances in our understanding of ion channel disorders. The highlights of these advances include a detailed delineation of the molecular mechanisms underlying inherited cardiac arrhythmias and the discovery that ion channel mutations can contribute to neural development and neurodegeneration.

Short- and long-term effects of amiodarone on the two components of cardiac delayed rectifier K(+) current.

BACKGROUND: Amiodarone is the most promising drug for the treatment of life-threatening tachyarrhythmias in patients with structural heart disease. The pharmacological effects of amiodarone on cardiac ion channels are complex and may differ for short-term and long-term administration. METHODS AND RESULTS: The delayed rectifier K(+) current (I(K)) of ventricular myocytes isolated from rabbit hearts was recorded with the whole-cell voltage-clamp technique. I(K) was separated into 2 components by use of specific blockers for either I(Ks) (chromanol 293B, 30 micromol/L) or I(Kr) (E-4031, 10 micromol/L). Short-term application of amiodarone caused a concentration-dependent decrease in I(Kr) with an IC(50) of 2.8 micromol/L (n=8) but only a minimal reduction in I(Ks). The short-term effects of amiodarone were also determined in Xenopus oocytes expressing the cloned human channels that conduct I(Kr) and I(Ks) (HERG and KvLQT1/minK). HERG current in oocytes was reduced by amiodarone (IC(50)=38 micromol/L), whereas KvLQT1/minK current was unaffected by 300 micromol/L amiodarone. To study the effects of long-term drug administration, rabbits were treated for 4 weeks with oral amiodarone (100 mg. kg(-1). d(-1)) before cell isolation. Long-term administration of amiodarone decreased I(K) to 55% (n=10) in control rabbits and altered the relative density of I(Kr) and I(Ks). The majority (92%) of current was I(Kr). mRNA levels of rabbit ERG,KVLQT1, and minK in left ventricular myocardium did not differ between control and long-term amiodarone. CONCLUSIONS: Amiodarone has differential effects on the 2 components of I(K), depending on the application period; short-term treatment inhibits primarily I(Kr), whereas long-term treatment reduces I(Ks).

Long-QT syndrome-associated missense mutations in the pore helix of the HERG potassium channel.

BACKGROUND: Mutations in the human ether-à-go-go-related gene (HERG) cause chromosome 7-linked long-QT syndrome (LQTS), an inherited disorder of cardiac repolarization that predisposes affected individuals to arrhythmia and sudden death. METHODS AND RESULTS: Here, we characterize the physiological consequences of 3 LQTS-associated missense mutations (V612L, T613M, and L615V) located in the pore helix of the HERG channel subunit. Mutant HERG subunits were heterologously expressed in Xenopus oocytes alone or in combination with wild-type HERG subunits. Two-microelectrode voltage-clamp techniques were used to record currents, and a single oocyte chemiluminescence assay was used to assay surface expression of epitope-tagged subunits. When expressed alone, V612L and T613M HERG subunits did not induce detectable currents, and L615V induced very small currents. Coexpression of mutant and wild-type HERG subunits caused a dominant-negative effect that varied for each mutation. CONCLUSIONS: These findings define the physiological consequences of mutations in HERG that cause LQTS and indicate the importance of the pore helix of HERG for normal channel function.

Inactivation of calcium channel current in the calf cardiac Purkinje fiber. Evidence for voltage- and calcium-mediated mechanisms.

We have studied the influence of divalent cations on Ca channel current in the calf cardiac Purkinje fiber to determine whether this current inactivates by voltage- or Ca-mediated mechanisms, or by a combination of the two. We measured the reversal (or zero current) potential of the current when Ba, Sr, or Ca were the permeant divalent cations and determined that depletion of charge carrier does not account for time-dependent relaxation of Ca channel current in these preparations. Inactivation of Ca channel current persists when Ba or Sr replaces Ca as the permeant divalent cation, but the voltage dependence of the rate of inactivation is markedly changed. This effect cannot be explained by changes in external surface charge. Instead, we interpret the results as evidence that inactivation is both voltage and Ca dependent. Inactivation of Sr or Ba currents reflects a voltage-dependent process. When Ca is the divalent charge carrier, an additional effect is observed: the rate of inactivation is increased as Ca enters during depolarizing pulses, perhaps because of an additional Ca-dependent mechanism.

Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents.

An envelope of tails test was used to show that the delayed rectifier K+ current (IK) of guinea pig ventricular myocytes results from the activation of two outward K+ currents. One current was specifically blocked by the benzenesulfonamide antiarrhythmic agent, E-4031 (IC50 = 397 nM). The drug-sensitive current, "IKr" exhibits prominent rectification and activates very rapidly relative to the slowly activating drug-insensitive current, "IKs." IKs was characterized by a delayed onset of activation that occurs over a voltage range typical of the classically described cardiac IK. Fully activated IKs, measured as tail current after 7.5-s test pulses, was 11.4 times larger than the fully activated IKr. IKr was also blocked by d-sotalol (100 microM), a less potent benzenesulfonamide Class III antiarrhythmic agent. The activation curve of IKr had a steep slope (+7.5 mV) and a negative half-point (-21.5 mV) relative to the activation curve of IKs (slope = +12.7 mV, half-point = +15.7 mV). The reversal potential (Erev) of IKr (-93 mV) was similar to EK (-94 mV for [K+]o = 4 mM), whereas Erev of IKs was -77 mV. The time constants for activation and deactivation of IKr made up a bell-shaped function of membrane potential, peaking between -30 and -40 mV (170 ms). The slope conductance of the linear portion of the fully activated IKr-V relation was 22.5 S/F. Inward rectification of this relation occurred at potentials greater than -50 mV, resulting in a voltage-dependent decrease in peak IKr at test potentials greater than 0 mV. Peak IKr at 0 mV averaged 0.8 pA/pF (n = 21). Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (225 ms) at membrane potentials (-20 to +20 mV) typical of the plateau phase of cardiac action potentials.

Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate.

Deactivation of voltage-gated potassium (K(+)) channels can slow or prevent the recovery from block by charged organic compounds, a phenomenon attributed to trapping of the compound within the inner vestibule by closure of the activation gate. Unbinding and exit from the channel vestibule of a positively charged organic compound should be favored by membrane hyperpolarization if not impeded by the closed gate. MK-499, a methanesulfonanilide compound, is a potent blocker (IC(50) = 32 nM) of HERG K(+) channels. This bulky compound (7 x 20 A) is positively charged at physiological pH. Recovery from block of HERG channels by MK-499 and other methanesulfonanilides is extremely slow (Carmeliet 1992; Ficker et al. 1998), suggesting a trapping mechanism. We used a mutant HERG (D540K) channel expressed in Xenopus oocytes to test the trapping hypothesis. D540K HERG has the unusual property of opening in response to hyperpolarization, in addition to relatively normal gating and channel opening in response to depolarization (Sanguinetti and Xu 1999). The hyperpolarization-activated state of HERG was characterized by long bursts of single channel reopening. Channel reopening allowed recovery from block by 2 microM MK-499 to occur with time constants of 10.5 and 52.7 s at -160 mV. In contrast, wild-type HERG channels opened only briefly after membrane hyperpolarization, and thus did not permit recovery from block by MK-499. These findings provide direct evidence that the mechanism of slow recovery from HERG channel block by methanesulfonanilides is due to trapping of the compound in the inner vestibule by closure of the activation gate. The ability of HERG channels to trap MK-499, despite its large size, suggests that the vestibule of this channel is larger than the well studied Shaker K(+) channel.

Voltage-dependent modulation of Ca channel current in heart cells by Bay K8644.

We have investigated the voltage-dependent effects of the dihydropyridine Bay K8644 on Ca channel currents in calf Purkinje fibers and enzymatically dispersed rat ventricular myocytes. Bay K8644 increases the apparent rate of inactivation of these currents, measured during depolarizing voltage pulses, and shifts both channel activation and inactivation in the hyperpolarizing direction. Consequently, currents measured after hyperpolarizing conditioning pulses are larger in the presence of drug compared with control conditions, but are smaller than control if they are measured after positive conditioning pulses. Most of our experimental observations on macroscopic currents can be explained by a single drug-induced change in one rate constant of a simple kinetic model. The rate constant change is consistent with results obtained by others with single channel recordings.

Fast inactivation causes rectification of the IKr channel.

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current-voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.

Modulation of potassium channels by antiarrhythmic and antihypertensive drugs.

Agents that modulate cardiac and smooth muscle K+ channels have stimulated considerable interest in recent years because of their therapeutic potential in a number of cardiovascular diseases. Foremost among these drugs are the so-called Class III antiarrhythmic agents, which act by prolonging cardiac action potentials, and K+ channel openers, which hyperpolarize and thereby relax smooth muscle cells. Many of the newly developed Class III antiarrhythmic agents probably act by specific block of one subtype of delayed rectifier K+ current, IKr, whereas other agents block more than one type of cardiac K+ current. Much controversy exists over the specific type of K+ channel (or channels) in smooth muscle that are activated by the K+ channel openers. Both groups of K+ channel modulators have great therapeutic promise, but the Class III antiarrhythmic agents may suffer from a side-effect that is directly linked to their specific mechanism of action.

Functional consequences of chloride channel gene (CLCN1) mutations causing myotonia congenita.

OBJECTIVE: To determine the functional consequences of missense mutations within the skeletal muscle chloride channel gene CLCN1 that cause myotonia congenita. BACKGROUND: Myotonia congenita is a genetic muscle disease associated with abnormalities in the skeletal muscle voltage-gated chloride (ClC-1) channel. In order to understand the molecular basis of this inherited disease, it is important to determine the physiologic consequences of mutations found in patients affected by it. METHODS: The authors used a mammalian cell (human embryonic kidney 293) expression system and the whole-cell voltage-clamp technique to functionally express and physiologically characterize five CLCN1 mutations. RESULTS: The I329T mutation shifted the voltage dependence of open probability of ClC-1 channels to the right by 192 mV, and the R338Q mutation shifted it to the right by 38 mV. In addition, the I329T ClC-1 channels deactivated to a lesser extent than normal at negative potentials. The V165G, F167L, and F413C ClC-1 channels also shifted the voltage dependence of open probability, but only by +14 to +20 mV. CONCLUSIONS: The functional consequences of these mutations form the physiologic argument that these are disease-causing mutations and could lead to myotonia congenita by impairing the ability of the skeletal muscle voltage-gated chloride channels to maintain normal muscle excitability. Understanding of genetic and physiologic defects may ultimately lead to better diagnosis and treatment of patients with myotonia congenita.

Molecular biology of K(+) channels and their role in cardiac arrhythmias.

The configuration of cardiac action potentials varies considerably from one region of the heart to another. These differences are caused by differential cellular expression of several types of K(+) channel genes. The channels encoded by these genes can be grouped into several classes depending on the stimulus that permits the channels to open and conduct potassium ions. K(+) channels are activated by changes in transmembrane voltage or binding of ligands. Voltage-gated channels are normally the most important players in determining the shape and duration of action potentials and include the delayed rectifiers and the transient outward potassium channels. Ligand-gated channels include those that probably have only minor roles in shaping repolarization under normal conditions but, when activated by extracellular acetylcholine or a decrease in the intracellular concentration of ATP, can substantially shorten action potential duration. Inward rectifier K(+) channels are unique in that they are basically stuck in the open state but can be blocked in a voltage-dependent manner by intracellular Mg(2+), Ca(2+), and polyamines. Other K(+) channels have been described that provide a small background leak conductance. Many of these cardiac K(+) channels have been cloned in the past decade, permitting detailed studies of the molecular basis of their function and facilitating the discovery of the molecular basis of several forms of congenital arrhythmias. Drugs that block cardiac K(+) channels and prolong action potential duration have been developed as antiarrhythmic agents. However, many of these same drugs, as well as other common medications that are structurally unrelated, can also cause long QT syndrome and induce ventricular arrhythmia.

Potassium channelopathies.

The molecular diversity of K(+)-selective channels far exceeds any other group of voltage- or ligand-gated channels, reflecting their early ancestral origin. This diversity is mirrored by the broad spectrum of physiological functions subserved by these proteins. Potassium channels modulate the resting potential and action potential duration of neurons, myocytes and endocrine cells and stabilize the membrane potential of excitable and nonexcitable cells. In addition to channel diversity, differential cellular expression of K+ channels determines the specific electrical responses to stimuli in a particular cell or tissue. This study reviews the recent genetic and physiological studies of congenital disorders caused by mutations in genes encoding K+ channels. These include the human disorders of episodic ataxia with myokymia, long QT syndrome and Bartter's syndrome, and weaver ataxia in mice. An understanding of the molecular basis of these diseases could facilitate the discovery and development of specific pharmacological therapies.

Stimulation rate modulates effects of the dihydropyridine CGP 28 392 on cardiac calcium-dependent action potentials.

Calcium (Ca2+)-dependent action potentials were recorded from 22 mM potassium (K+)-depolarized guinea-pig papillary muscle at several different pacing frequencies in the absence and presence of CGP 28 392 (10 microM), a Ca2+ channel agonist. The maximum upstroke velocity (Vmax) of the slow response action potential was measured to determine relative changes in Ca2+ current as a function of pacing frequency. CGP 28 392 increased Vmax more than two fold at low rates of stimulation (1 or 12 pulses min-1), but had no significant effect on Vmax during rapid pulsing (200 pulses min-1). The enhancement of Vmax was dependent upon extracellular [K+]. Increasing extracellular [K+] from 22 mM to 27 mM suppressed the frequency-dependent agonist effects and increased the antagonist effects on Vmax. These results indicate that CGP 28 392 is a partial Ca2+-channel agonist and suggest that its effects on Ca2+ current are voltage-dependent.

Dysfunction of delayed rectifier potassium channels in an inherited cardiac arrhythmia.

The rapid (IKr) and slow (IKs) delayed rectifier K+ currents are key regulators of cardiac repolarization. HERG encodes the Kr channel, and KVLQT1 and hminK encode subunits that coassemble to form Ks channels. Mutations in any one of these genes cause Romano-Ward syndrome, an autosomal dominant form of long QT syndrome (LQT). Mutations in KVLQT1 and HERG are the most common cause of LQT. Not all missense mutations of HERG or KVLQT1 have the same effect on K+ channel function. Most mutations result in a dominant-negative effect, but the severity of the resulting phenotype varies widely, as judged by reduction of current induced by coexpression of wild-type and mutant subunits in heterologous expression systems. Mutations in hminK (S74L, D76N) reduce IKs by shifting the voltage dependence of activation and accelerating channel deactivation. A recessive form of LQT is caused by mutations in either KVLQT1 or hminK. The functional consequences of mutations in delayed rectifier K+ channel subunits are delayed cardiac repolarization, lengthened QT interval, and an increased risk of torsade de pointes and sudden death.

Long QT syndrome: ionic basis and arrhythmia mechanism in long QT syndrome type 1.

Long QT syndrome type 1 (LQT1) causes torsades de pointes arrhythmia, ventricular fibrillation, and sudden death. It usually is inherited as an autosomal dominant trait (Romano-Ward syndrome). The primary defect in LQT1 is a mutation in KVLQT1, a gene that encodes the pore-forming alpha-subunit of a K+ channel. KvLQT1 alpha-subunits coassemble with minK beta-subunits to form channels that conduct the slow delayed rectifier K+ current (I(Ks)) in the heart. Recessive mutations in KVLQT1 cause Jervell and Lange-Nielsen syndrome, which is characterized by more severe arrhythmias and congenital neural deafness. Heterologous expression studies demonstrated that mutations in KVLQT1 reduce I(Ks) by causing loss of channel function, altered channel gating, and/or a dominant-negative effect. It remains to be proven that an understanding of the molecular basis of LQT1 will lead to more effective therapy.

Voltage-dependent block of calcium channel current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel antagonists.

We have investigated the mechanisms of blockade of calcium channel current by the dihydropyridines, e.g. nisoldipine, nitrendipine, and nicardipine. Membrane current was recorded in isolated calf Purkinje fibers using a two-microelectrode voltage-clamp technique, and voltage protocols were designed to identify voltage- and use-dependent block by these compounds systematically. Our results show that calcium channel blockade by dihydropyridine derivatives is strongly modulated by membrane potential. Block is more pronounced when current is measured from depolarized holding potentials, but in contrast to verapamil, this voltage-dependent block occurs in the absence of repetitive depolarizations. Use-dependent block by dihydropyridines is observed at pulse frequencies greater than 1 Hz. Our results suggest that dihydropyridines bind preferentially to the inactivated state of the calcium channel, and that the development of use-dependent block is related to the ionization constants of the compounds. Furthermore, binding is approximately one thousand times stronger to inactivated channels than to resting channels. This state-dependent difference in binding affinities may account for the previously reported contrast between electrophysiological and binding data for these compounds.