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Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated [Na.sup.+] channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the [alpha] subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH ter-minus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the [Na.sup.+] channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process. KEY WORDS: inactivation * long QT syndrome * sodium channel * structure * heart

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, ~50% neutral at pH 7.4) have similar structures but markedly different effects on [Na.sup.+] channel activity. Both drugs cause well-characterized use-dependent block (UDB) of [Na.sub.+] channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of [Na.sup.+] channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na+ channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac [Na.sup.+] channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules. KEY WORDS: electrophysiology * local anesthetics * flecainide * arrhythmia * pharmacology

[Na.sup.+] channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the [alpha] subunit of the cardiac voltage-gated [Na.sup.+] channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the [Na.sup.+] channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.

An analysis of the interaction between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells revealed that the current through the ATP-sensitive K channel (I-KATP) is blocked by extracellularly-applied Co2+, Cd2+ and Zn2+ in whole-cell patch clamp recording conditions. A few divalent cations inhibit current through I-KATP channels by binding to regions outside the transmembrane electric field. The block interdependence of internal ATP and external divalent cation binding reveal that a modulatory mechanism is possible.

Investigates the interactions between extracellular divalent cations and the adenosine triphosphatase-sensitive potassium channel. Blocking of current by extracellularly applied cobalt, cadmium and zinc cations; Concentrations; Whole-cell patch clamping conditions; Dependency of blockage on intracellular ATP concentrations; Conformational change of channel protein.

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, ∼50% neutral at pH 7.4) have similar structures but markedly different effects on Na[sup +] channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na[sup +] channels due to stabilization of the inactivated state, but fiecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na[sup +] channel blockade, we developed two fiecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na[sup +] channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of fiecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na[sup +] channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by... [ABSTRACT FROM AUTHOR]

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na+channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na+channel current (INAL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of (INAL)such that increasing the pacing rate markedly reduces (INAL) and, in addition, increases its inhibition by the Na+channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband's iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens. [ABSTRACT FROM AUTHOR]

Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the α subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process. [ABSTRACT FROM AUTHOR]

Presents a study that determined what disease-modified channel properties underlie distinct responses to the sodium ion (Na[sup+]) channel blocker flecainide in Brugada syndrome (BrS) and LQT-3, a variant of the Long QT syndrome mutations. Analysis of the molecular determinants of flecainide block; Influence of inherited mutations on the voltage dependence of flecainide use-dependent block; Impact of flecainide on Na[sup+] channel availability.