RESUMEN
Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels.
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Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.
Asunto(s)
Potenciales de Acción , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Células COS , Diferenciación Celular , Línea Celular , Canal de Potasio ERG1/metabolismo , Fenómenos Electrofisiológicos , Expresión Génica , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Canal de Potasio KCNQ1/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN5A, the molecular mechanisms underlying this condition are still largely unknown. METHODS AND RESULTS: We combined whole-exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB2, which encodes the voltage-gated K(+) channel ß2-subunit (Kvß2-R12Q). Kvß2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K(+) channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB2 variants by using cellular electrophysiology and biochemistry. Patch-clamp experiments performed in COS-7 cells expressing both Kv4.3 and Kvß2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvß2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvß2-R12Q were significantly increased in comparison with wild-type Kvß2. CONCLUSIONS: Altogether, our results indicate that Kvß2 dysfunction can contribute to the Brugada electrocardiographic pattern.
Asunto(s)
Síndrome de Brugada/genética , Mutación con Ganancia de Función/genética , Canales de Potasio con Entrada de Voltaje/genética , Electrocardiografía , Femenino , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Masculino , Canal de Sodio Activado por Voltaje NAV1.5/genética , Linaje , Polimorfismo de Nucleótido Simple/genética , Canales de Potasio de la Superfamilia Shaker , Canales de Potasio Shal/genética , Secuenciación del ExomaRESUMEN
BACKGROUND: Human genetically inherited cardiac diseases have been studied mainly in heterologous systems or animal models, independent of patients' genetic backgrounds. Because sources of human cardiomyocytes (CMs) are extremely limited, the use of urine samples to generate induced pluripotent stem cell-derived CMs would be a noninvasive method to identify cardiac dysfunctions that lead to pathologies within patients' specific genetic backgrounds. The objective was to validate the use of CMs differentiated from urine-derived human induced pluripotent stem (UhiPS) cells as a new cellular model for studying patients' specific arrhythmia mechanisms. METHODS AND RESULTS: Cells obtained from urine samples of a patient with long QT syndrome who harbored the HERG A561P gene mutation and his asymptomatic noncarrier mother were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into CMs using the matrix sandwich method.UhiPS-CMs showed proper expression of atrial and ventricular myofilament proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials recorded using high-throughput optical and patch-clamp techniques. Comparison of HERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in reduced delayed rectifier K(+) current (IKr). This phenotype gave rise to action potential prolongation and arrhythmias. CONCLUSIONS: UhiPS cells from patients carrying ion channel mutations can be used as novel tools to differentiate functional CMs that recapitulate cardiac arrhythmia phenotypes.
Asunto(s)
Diferenciación Celular , Síndrome de QT Prolongado/orina , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismo , Medicina de Precisión/métodos , Potenciales de Acción , Técnicas de Cultivo de Célula , Células Cultivadas , Técnicas de Reprogramación Celular , Canal de Potasio ERG1 , Electrocardiografía , Canales de Potasio Éter-A-Go-Go/genética , Femenino , Predisposición Genética a la Enfermedad , Ensayos Analíticos de Alto Rendimiento , Humanos , Síndrome de QT Prolongado/genética , Síndrome de QT Prolongado/patología , Masculino , Persona de Mediana Edad , Mutación Missense , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Fenotipo , Células Madre Pluripotentes/patología , Orina/citología , Adulto JovenRESUMEN
The human ether-á-go-go-related gene (hERG) K(+) channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q(1) and Q(2), with V(1/2)'s of -55.7 (equivalent charge, z = 1.60) and -54.2 mV (z = 1.30), respectively, with the Q(2) charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q(1) and Q(2), decreasing to 4.3 ms for Q(2) at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V(1/2) of -64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q(1) and Q(2) charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Activación del Canal Iónico , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Línea Celular , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/química , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Potenciales de la Membrana , Mesilatos/farmacología , Datos de Secuencia Molecular , Mutación Missense , Estructura Terciaria de Proteína , Electricidad EstáticaRESUMEN
The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG(+)) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs(+), displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K(+), suggesting that K(+) ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG(+) ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K(+), allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.
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Activación del Canal Iónico , Canal de Potasio Kv.1.2/fisiología , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Cationes/metabolismo , Línea Celular , Cesio/farmacología , Humanos , Espacio Intracelular/química , Activación del Canal Iónico/efectos de los fármacos , Canal de Potasio Kv.1.2/química , Canal de Potasio Kv.1.2/metabolismo , Meglumina/farmacología , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Potasio/metabolismo , Estructura Terciaria de Proteína , Tetraetilamonio/farmacologíaRESUMEN
Phosphatidylinositol (4,5)-bisphosphate (PIP(2)) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP(2) on the open state stabilization. A similar effect of PIP(2) on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K(+) channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP(2) effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP(2) exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations.
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Activación del Canal Iónico/fisiología , Canal de Potasio KCNQ1/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismo , Animales , Células COS , Chlorocebus aethiops , Canal de Potasio KCNQ1/genética , Fosfatidilinositol 4,5-Difosfato/genética , Canales de Potasio de la Superfamilia Shaker/genética , XenopusRESUMEN
Voltage-gated potassium (Kv) channels are tetramers, each subunit presenting six transmembrane segments (S1-S6), with each S1-S4 segments forming a voltage-sensing domain (VSD) and the four S5-S6 forming both the conduction pathway and its gate. S4 segments control the opening of the intracellular activation gate in response to changes in membrane potential. Crystal structures of several voltage-gated ion channels in combination with biophysical and mutagenesis studies highlighted the critical role of the S4-S5 linker (S4S5(L)) and of the S6 C-terminal part (S6(T)) in the coupling between the VSD and the activation gate. Several mechanisms have been proposed to describe the coupling at a molecular scale. This review summarizes the mechanisms suggested for various voltage-gated ion channels, including a mechanism that we described for KCNQ1, in which S4S5(L) is acting like a ligand binding to S6(T) to stabilize the channel in a closed state. As discussed in this review, this mechanism may explain the reverse response to depolarization in HCN-like channels. As opposed to S4S5(L), the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP(2)), stabilizes KCNQ1 channel in an open state. Many other ion channels (not only voltage-gated) require PIP(2) to function properly, confirming its crucial importance as an ion channel cofactor. This is highlighted in cases in which an altered regulation of ion channels by PIP(2) leads to channelopathies, as observed for KCNQ1. This review summarizes the state of the art on the two regulatory mechanisms that are critical for KCNQ1 and other voltage-gated channels function (PIP(2) and S4S5(L)), and assesses their potential physiological and pathophysiological roles.
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G628S is a mutation in the signature sequence that forms the selectivity filter of the human ether-a-go-go-related gene (hERG) channel (GFG) and is associated with long-QT2 syndrome. G628S channels are known to have a dominant-negative effect on hERG currents, and the mutant is therefore thought to be nonfunctional. This study aims to assess the physiological mechanism that prevents the surface-expressing G628S channels from conducting ions. We used voltage-clamp fluorimetry along with two-microelectrode voltage clamping in Xenopus oocytes to confirm that the channels express well at the surface, and to show that they are actually functional, with activation kinetics comparable to that of wild-type, and that the mutation leads to a reduced selectivity to potassium. Although ionic currents are not detected in physiological solutions, removing extracellular K(+) results in the appearance of an inward Na(+)-dependent current. Using whole-cell patch clamp in mammalian transfected cells, we demonstrate that the G628S channels conduct Na(+), but that this can be blocked by both intracellular and higher-than-physiological extracellular K(+). Using solutions devoid of K(+) allows the appearance of nA-sized Na(+) currents with activation and inactivation gating analogous to wild-type channels. The G628S channels are functionally conducting but are normally blocked by intracellular K(+).
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Canales de Potasio Éter-A-Go-Go/química , Canales de Potasio Éter-A-Go-Go/metabolismo , Activación del Canal Iónico/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Canal de Potasio ERG1 , Conductividad Eléctrica , Canales de Potasio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Oocitos/metabolismo , Permeabilidad , Conformación Proteica , Sodio/metabolismo , Xenopus/genéticaRESUMEN
Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil.
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Moduladores del GABA/farmacología , Insecticidas/farmacología , Sistemas Neurosecretores/efectos de los fármacos , Periplaneta/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Receptores de GABA/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Relación Dosis-Respuesta a Droga , Activadores de Enzimas/farmacología , Masculino , Potenciales de la Membrana , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/enzimología , Técnicas de Placa-Clamp , Periplaneta/enzimología , Fosforilación , Proteína Quinasa C/metabolismo , Receptores de GABA/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channel's S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. OBJECTIVE: This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. METHODS: Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. RESULTS: Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. CONCLUSION: The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.
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Canales de Potasio Éter-A-Go-Go/genética , Síndrome de QT Prolongado/genética , ADN Complementario/genética , Canal de Potasio ERG1 , Fluorometría , Células HEK293 , Humanos , Síndrome de QT Prolongado/congénito , Mutación , Técnicas de Placa-Clamp/métodos , Fenetilaminas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Transporte de Proteínas , Sulfonamidas/farmacología , TransfecciónRESUMEN
The K(2P) potassium channels are responsible for the background conductance observed in several tissues. Their ubiquitous localization and thus their potential implications in diseases have led to increased research on these channels over the last few years. In this review, we outline different aspects of the research on K(2P) channels and highlight some of the latest discoveries in this area. We focus on research into K(2P) channels as potential therapeutic targets in ischemia/hypoxia, depression, memory disorders, pain, cardiovascular disease and disorders of the immune system. We address the challenge of developing novel pharmacological compounds to target these channels. We also discuss the regulation of expression of the K(2P) gene in health and disease, as well as the value of assessing the expression of K(2P) channels as potential biomarkers of disease.
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Enfermedades Cardiovasculares/metabolismo , Infertilidad Masculina/metabolismo , Trastornos Mentales/metabolismo , Neoplasias/metabolismo , Bloqueadores de los Canales de Potasio/uso terapéutico , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Animales , Sistemas de Liberación de Medicamentos/métodos , Dosificación de Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/genética , Canales de Potasio/metabolismoRESUMEN
BACKGROUND: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. METHODS AND FINDINGS: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2)) of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON), with V((1/2)) (,1) = -37.8+/-1.7 mV, and V((1/2)) (,2) = 43.5+/-7.9 mV. The first phase, V((1/2)) (,1), was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2)) = -18.3+/-1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2)) = -34.4+/-1.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2)) = -20.6+/-1.2, k = 11.4 mV) and L520C quenching during depolarization (V((1/2)) = -26.8+/-1.0, k = 13.3 mV) matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing. CONCLUSION: THE DATA INDICATE: 1) that rapid environmental changes occur at the outer end of S4 in hERG channels that underlie channel activation gating, and 2) that secondary slower changes reflect channel pore opening during sustained depolarizations, and channel closing upon repolarization. 3) No direct evidence was obtained of conformational changes related to inactivation from fluorophores attached at the outer end of S4.