Channel Gating Parameters Research Articles

Molecular Basis of Mexiletine Response Variability in Sodium Channels with Long QT Mutations

Background: Long-QT syndrome type-3 (LQT3) is a life-threatening disease caused by mutations in the cardiac sodium channel (NaV1.5). The class-Ib anti-arrhythmic mexiletine was proposed as an effective therapy for LQT3. However, clinical studies showed that LQT3 variants have distinct sensitivities to mexiletine. We hypothesized that altered voltage-sensing domain (VSD) function determines mutation-dependent mexiletine blockade. Methods: NaV1.5 contains four domains (DI-DIV), each with a VSD. We previously created four channel constructs that can track each VSD's conformation changes when tethered with a fluorophore. VSD-tracking fluorescence and ionic currents were recorded simultaneously for WT and LQT3-linked channels with or without mexiletine. Results: Mexiletine blockade of WT channel stabilized the DIII-VSD activated conformation. Among 14 common LQT mutations, 10 showed varied DIII-VSD activation voltage-dependence, despite distal locations of some, e.g. R1626P(DIV-VSD) and E1784K(C-terminus). DIII-VSD activation shifts correlate strongly with tonic block by mexiletine(R=0.91), suggesting that the DIII-VSD conformation determines mexiletine blockade at the cardiomyocyte resting potential. Decoupling the DIII-VSD from the pore eliminated mutation-specific mexiletine sensitivity, suggesting that the activated DIII-VSD facilitates mexiletine block by promoting pro-binding conformations within the DIII pore. Unlike WT, mexiletine blockade of the inactivation-impaired channel (F1486Q) did not shift the DIII-VSD, implying that during the action potential plateau, the DIII-VSD and inactivation gate coordinate to regulate mexiletine blockade. To predict use-dependent block (UDB) and QT-interval shortening (ΔQT) from channel gating parameters, we applied a machine learning approach, partial least-square regression(PLSR). Our PLSR model showed that changes in DIII-VSD activation, steady-state inactivation and slow recovery from inactivation parameters are strongly predictive of mexiletine-linked changes in UDB and ΔQT (RUDB=0.92, RΔQT=0.82). Conclusion: Traditional anti-arrhythmic block models have focused on the role of channel inactivation. We have revised this model to include an essential role of the DIII-VSD.

The Sh3-Binding Domain of Kv1.3 Channels is Required for their Cortactin-Conveyed Coupling to Actin

Kv1.3 channels of T lymphocytes play an important role in several physiological functions like activation or migration, which are accompanied by the redistribution of channels in the cell membrane. Both processes are characterized by F-actin polymerization indicating the involvement of the actin cytoskeleton in the associated channel's redistribution. Several studies reported that Kv1.x channels are linked to or co-localize with various adapter proteins which also possess actin-binding domains: hDlg1 via the PDZ-binding domain and cortactin through either the cortactin- or the SH3-binding site. In this study we investigated the molecular requirements for Kv1.3 channel lateral membrane diffusion.FLAG/EGFP-tagged Kv1.3 wild-type or mutant constructs were expressed in HEK-293 cells. We designed C-terminal truncated (without (delta1) or with (delta2) cortactin binding sequence, and both without SH3; with cortactin and SH3 binding motif (delta3)) and few-amino-acid Kv1.3 mutants (deltaSH3/deltaPDZ: SH3/PDZ domain ruptured). Biophysical properties of each phenotype were evaluated with patch-clamping. Interaction/co-localization between channel and adaptor proteins was studied with co-immunoprecipitation/confocal microscopy. Lateral mobility of Kv1.3 was assessed with FRAP and defined by the channel's mobile fraction (Mf).Parameters of channel gating for mutants were indistinguishable from those of wild-type Kv1.3. Confocal microscopy images and co-immunoprecipitation experiments demonstrated that cortactin and Kv1.3 interact in HEK-293 cells. Furthermore, wild-type, delta3 and deltaPDZ mutants have reduced Mf upon stimulation of F-actin polymerization/stabilization by jasplakinolide (unlike delta1, delta2 and deltaSH3 constructs). When cortactin was knocked down by shRNA, exposure to jasplakinolide induced no decrease in Mf of wild-type Kv1.3 channels. These findings point out that cortactin serves as a bridge between Kv1.3 and the actin meshwork via the channel's SH3-binding domain, and can regulate mobility/immobility of Kv1.3 channels. (NIH 2R01CA095286).

Voltage-Gated Ion Channel Dysfunction Precedes Cardiomyopathy Development in the Dystrophic Heart

BackgroundDuchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology.Methodology/Principal FindingsTo address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged.Conclusions/SignificanceIon channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers.

Temporal lobe epilepsy induces intrinsic alterations in Na channel gating in layer II medial entorhinal cortex neurons

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy involving the limbic structures of the temporal lobe. Layer II neurons of the entorhinal cortex (EC) form the major excitatory input into the hippocampus via the perforant path and consist of non-stellate and stellate neurons. These neurons are spared and hyper-excitable in TLE. The basis for the hyper-excitability is likely multifactorial and may include alterations in intrinsic properties. In a rat model of TLE, medial EC (mEC) non-stellate and stellate neurons had significantly higher action potential (AP) firing frequencies than in control. The increase remained in the presence of synaptic blockers, suggesting intrinsic mechanisms. Since sodium (Na) channels play a critical role in AP generation and conduction we sought to determine if Na channel gating parameters and expression levels were altered in TLE. Na channel currents recorded from isolated mEC TLE neurons revealed increased Na channel conductances, depolarizing shifts in inactivation parameters and larger persistent ( I NaP) and resurgent ( I NaR) Na currents. Immunofluorescence experiments revealed increased staining of Na v1.6 within the axon initial segment and Na v1.2 within the cell bodies of mEC TLE neurons. These studies provide support for additional intrinsic alterations within mEC layer II neurons in TLE and implicate alterations in Na channel activity and expression, in part, for establishing the profound increase in intrinsic membrane excitability of mEC layer II neurons in TLE. These intrinsic changes, together with changes in the synaptic network, could support seizure activity in TLE.

A Virtual Hair Cell, II: Evaluation of Mechanoelectric Transduction Parameters

The virtual hair cell we have proposed utilizes a set of parameters related to its mechanoelectric transduction. In this work, we observed the effect of such channel gating parameters as the gating threshold, critical tension, resting tension, and Ca 2+ concentration. The gating threshold is the difference between the resting and channel opening tension exerted by the tip link assembly on the channel. The critical tension is the tension in the tip link assembly over which the channel cannot close despite Ca 2+ binding. Our results show that 1), the gating threshold dominated the initial sensitivity of the hair cell; 2), the critical tension minimally affects the peak response, ( I), but considerably affects the time course of response, I( t), and the force-displacement, F-X, relationship; and 3), higher intracellular [Ca 2+] resulted in a smaller fast adaptation time constant. Based on the simulation results we suggest a role of the resting tension: to help overcome the viscous drag of the hair bundle during the oscillatory movement of the bundle. Also we observed the three-dimensional bundle effect on the hair cell response by varying the number of cilia forced. These varying forcing conditions affected the hair cell response.

Characterization of three myotonia-associated mutations of the CLCN1 chloride channel gene via heterologous expression.

Two novel mutations of the human CLCN1 chloride channel gene, c.592C>G (p.L198V) and c.2255A>G (p.K752R), are described, occurring coincidentally in the one myotonic patient. These individual mutations and a construct with both mutations in the one cDNA were transcribed and expressed in Xenopus oocytes where channel gating parameters were extracted from chloride currents recorded under voltage clamp. We found that the p.L198V mutation has its major effects on the common (or slow) gate of the chloride channel, as do other dominant ClC-1 mutations, and may therefore be causative of the patient's symptoms (when co-expressed with wild-type human ClC-1, the p.L198V mutation exerts a dominant negative effect on common gating) but the p.K752R mutation appears to be innocuous and may be a benign polymorphism. A third mutant, the recently described c.2795C>T (p.P932L), was expressed in HEK 293 cells. Despite the severity of the disease associated with this mutation, chloride currents in cells expressing p.P932L were not significantly different from those of cells expressing wild-type ClC-1.

Solution Structure and Function of the “Tandem Inactivation Domain” of the Neuronal A-type Potassium Channel Kv1.4

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Single Channel Analysis of the Regulation of GIRK1/GIRK4 Channels by Protein Phosphorylation

G-Protein activated, inwardly rectifying potassium channels (GIRKs) are important effectors of G-protein β/ γ-subunits, playing essential roles in the humoral regulation of cardiac activity and also in higher brain functions. G-protein activation of channels of the GIRK1/GIRK4 heterooligomeric composition is controlled via phosphorylation by cyclic AMP dependent protein kinase (PKA) and dephosphorylation by protein phosphatase 2A (PP 2A). To study the molecular mechanism of this unprecedented example of G-protein effector regulation, single channel recordings were performed on isolated patches of plasma membranes of Xenopus laevis oocytes. Our study shows that: ( i) The open probability ( P o) of GIRK1/GIRK4 channels, stimulated by coexpressed m 2-receptors, was significantly increased upon addition of the catalytic subunit of PKA to the cytosolic face of an isolated membrane patch. ( ii) At moderate concentrations of recombinant G β1/ γ2 , used to activate the channel, P o was significantly reduced in patches treated with PP 2A, when compared to patches with PKA-cs. ( iii) Several single channel gating parameters, including modal gating behavior, were significantly different between phosphorylated and dephosphorylated channels, indicating different gating behavior between the two forms of the protein. Most of these changes were, however, not responsible for the marked difference in P o at moderate G-protein concentrations. ( iv) An increase of the frequency of openings ( f o) and a reduction of dwell time duration of the channel in the long-lasting C 5 state was responsible for facilitation of GIRK1/GIRK4 channels by protein phosphorylation. Dephosphorylation by PP 2A led to an increase of G β1/ γ2 concentration required for full activation of the channel and hence to a reduction of the apparent affinity of GIRK1/GIRK4 for G β1/ γ2 . ( v) Although possibly not directly the target of protein phosphorylation/dephosphorylation, the last 20 C-terminal amino acids of the GIRK1 subunit are required for the reduction of apparent affinity for the G-protein by PP 2A, indicating that they constitute an essential part of the off-switch.

Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel

The effect of the trivalent cation lanthanum (La 3+) on voltage-dependent gating of a cloned mammalian neuronal Kv1.1 potassium channel was studied under whole-cell voltage-clamp conditions in oocytes of Xenopus laevis. La 3+ (100 μM) was found to decrease the potassium currents at all test potentials and to shift the midpoint of the fraction open channels/membrane voltage curve by approximately +20 mV. The opening and closing time constants of Kv1.1 channels were empirically fitted with a 4th power Hodgkin-Huxley formalism, or with mono- and multi-exponentials. It was found that La 3+ slowed down the kinetics of activation, speeded up those of deactivation, and shifted the opening kinetics by approximately +60 mV. Interestingly, all these parameters of channel gating were not affected equally by La 3+. Furthermore, amplitudes of the inward tail currents evoked at potentials more negative than the potassium equilibrium potential ( E K + ) were more strongly inhibited by La than those of the outward tail currents evoked at potentials more positive than E K + . This suggests voltage-dependent block and binding of La 3+ to the Kv1.1 channel protein. We conclude that these actions cannot be explained in terms of surface charge considerations alone. Our results provide evidence for a direct interaction with the potassium channel protein, shedding new light on the mechanism of action of this lanthanide.

Stationarity of sodium channel gating kinetics in excised patches from neuroblastoma N1E 115

Na channel gating parameters in a number of preparations are translated along the voltage axis in excised patches compared to cell attached or whole cell recording. The aim of this study is to determine whether these changes in gating behavior continue over an extended period or, rather, develop rapidly on excision with stationary kinetics thereafter. Average currents were constructed from single-channel records from neuroblastoma N1E 115 at various times after excision, excluding the first 5 min, in eight inside-out excised patches. Single exponentials were fitted to the current decay of the average records, and the mean time constant for each patch was determined. Values were plotted as the percentage difference from these means for each patch against time from excision. Collected results show no obvious trend in values from 5 min to 2 h. Kinetics are stationary, and shifts in Na channel gating parameters along the voltage axis seen in excised as compared to whole cell configuration in neuroblastoma must be complete by the first few minutes after excision. Raising the internal Na concentration reduced the single channel current amplitude, confirming that these are Na channels.

Anaesthetic modulation of nicotinic ion channel kinetics in bovine chromaffin cells

1. We have investigated the action of the anaesthetics methoxyflurane, methohexitone and etomidate on the nicotinic acetylcholine receptor channel of bovine adrenal chromaffin cells using the whole cell patch clamp technique. 2. Spectral analysis of macroscopic currents evoked by 25 microM carbachol revealed that each of the agents tested reduced the lifetime of the channel open state in a dose-dependent manner. The whole cell current was inhibited in a concentration-dependent fashion by each agent. 3. Channel gating parameters were calculated from single channel studies and the results used to test models explaining the modulation of nicotinic acetylcholine receptor channels by anaesthetics. 4. Each of the agents studied reduced the mean channel open time in a concentration-dependent manner. Anaesthetic concentrations reducing mean open time by 50% were: 370 microM methoxyflurane, 30 microM methohexitone or 23 microM etomidate. 5. Methohexitone and etomidate produced an increase in the number of brief closures within bursts, while no such increase was observed with methoxyflurane. Despite these inter-burst gaps, mean burst length was reduced by each of the agents tested. 6. It is concluded that a simple sequential blocking model fails to account for the action of these anaesthetics. An extended model, in which blocked channels can close, may be applicable.

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.

The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.

Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes.

The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKH of 5.10. The reduction in single channel current amplitude by protons is voltage independent between -70 and -20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h infinity) curve, the time constant for macroscopic current inactivation (tau h), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5 the midpoint of the h infinity curve shifts from -107.6 +/- 2.6 mV (mean +/- SD, n = 16) to -94.3 +/- 1.9 mV (n = 3, P less than 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h infinity curve with protons, yielding an estimate for total negative surface charge density of -1e/490 A2 and a pKH for proton binding of 5.16. By reducing external surface Na+ concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na+ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating.

Competitive blockage of the sodium channel by intracellular magnesium ions in central mammalian neurones.

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mgi2+, interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mgi2+. This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mgi2+ leads ot a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mgi2+ is decreased upon increasing intracellular or extracellular Na+ concentration, which suggests that Mgi2+ interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mgi2+ for the sodium channel has been calculated to be 8.65 +/- 1.51 mM, with its binding site located at 0.26 +/- 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Nai+, which is 83.76 +/- 7.60 mM with its binding site located at 0.75 +/- 0.23. The low dissociation constant of Mgi2+ reflects its high affinity for the sodium channel.