Current Inactivation Kinetics Research Articles

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navβ3 to Navβ4. Dynamic clamping and dialysis of Navβ4’s C-terminal peptide into juvenile RA neurons provide evidence that Navβ4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Characterization and modelling of the abnormal electrophysiological response under beta-adrenergic stimulation in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Introduction Hypertrophic Cardiomyopathy (HCM) is the most common inheritable heart pathology and the main cause of sudden cardiac death in young adults. HCM patients often present an enhanced arrhythmogenicity that can lead to lethal arrhythmias, especially during exercise. Recent studies have shown an abnormal response of HCM myocytes to β-adrenergic stimulation (β-ARS), with prolongation of their action potential duration (APD). The mechanisms underlying this aberrant response to sympathetic stimulation remain unknown. Purpose To investigate the key ionic mechanisms underlying the HCM abnormal response to β-ARS using human-based experimental and computational methodologies. Methods Experimental ionic currents, action potential and calcium transient were recorded in human adult cardiomyocytes from control and HCM patients. Isoproterenol (10-7 mol/L) was used to elicit β-ARS. Whole-cell ruptured patch voltage clamp experiments were conducted to characterise L-type calcium and potassium currents, with recordings performed before and after 3 min of drug exposure. The latest models of human ventricular electrophysiology and beta-adrenergic receptor signalling were integrated and calibrated using the human measured data. Simulations under isoproterenol were performed to quantify the effects of β-ARS on the action potential and calcium transient. The role of the main ion currents affected by β-ARS and by HCM remodelling was evaluated. Results In vitro, isoproterenol shortened APD (-16 ± 3%) in control, while prolonging APD in HCM myocytes (+23 ± 8%). Analysis of the measured data indicated two possible mechanisms contributing to APD prolongation in HCM myocytes. Firstly, a protracted L-type calcium current, presenting slower inactivation kinetics in HCM compared to control. The relative increase of potassium currents under β-ARS was also lower in HCM myocytes. The developed in silico models of β-ARS replicated the behaviour observed in the experimental data, based on slower L-type calcium current inactivation kinetics and a smaller increase of potassium currents in HCM. In absence of β-ARS, simulated HCM cardiomyocytes exhibited prolonged APD compared to control (525 ± 88 vs 281 ± 56 ms, p < 0.001). Under β-ARS, APD in control was reduced (-16.46%), whereas APD was prolonged in HCM (+11.63%). Further analysis showed that the reduction of the potassium currents increment under β-ARS was the main cause of the APD prolongation in HCM myocytes, with L-type calcium inactivation minimally contributing to APD prolongation. Conclusions In this study we assessed the effects of β-ARS on ion currents and APD in control and HCM myocytes. Our modelling results suggest that the increase of potassium repolarising currents under β-ARS is greatly reduced in HCM cardiomyocytes, being the main mechanism underlying their APD prolongation. This APD prolongation may have severe consequences in HCM patients, increasing the risk of exercise-induced arrhythmias. Abstract Figure.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca(2+) channel (CaV2.1α1). Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Functional analysis of the K1336E mutation revealed a reduction of Ca(2+) current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca(2+) channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

Theoretical study of L‐type Ca2+ current inactivation kinetics during action potential repolarization and early afterdepolarizations

Sarcoplasmic reticulum (SR) Ca(2+) release mediates excitation–contraction coupling (ECC) in cardiac myocytes. It is triggered upon membrane depolarization by entry of Ca(2+) via L-type Ca(2+) channels (LTCCs), which undergo both voltage- and Ca(2+)-dependent inactivation (VDI and CDI, respectively). We developed improved models of L-type Ca(2+) current and SR Ca(2+) release within the framework of the Shannon-Bers rabbit ventricular action potential (AP) model. The formulation of SR Ca(2+) release was modified to reproduce high ECC gain at negative membrane voltages. An existing LTCC model was extended to reflect more faithfully contributions of CDI and VDI to total inactivation. Ba(2+) current inactivation included an ion-dependent component (albeit small compared with CDI), in addition to pure VDI. Under physiological conditions (during an AP) LTCC inactivates predominantly via CDI, which is controlled mostly by SR Ca(2+) release during the initial AP phase, but by Ca(2+) through LTCCs for the remaining part. Simulations of decreased CDI or K(+) channel block predicted the occurrence of early and delayed after depolarizations. Our model accurately describes ECC and allows dissection of the relative contributions of different Ca(2+) sources to total CDI, and the relative roles of CDI and VDI, during normal and abnormal repolarization.

Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).

Role of the Putative Glycines Hinge of Cav3.3 Channels

Voltage-gated ion channels (VGIC) are proteins that form transmembrane pores in cell membranes. Based on the crystal structure of an open potassium channel (KV) that reveals the bending of a glycine residue in the inner helix that lines the pore (M2 or S6), and the conserved pore sequences in many members of the VGIC superfamily, is thought that the opening mechanism might be quite similar within the family. In fact, this has been demonstrated for a bacterial sodium channel (NaV). However, in LVA calcium channels, the mid-S6 hinge glycine residue is present only in IS6 and IIS6, suggesting a likely different opening mechanism. Here, we explored this possibility in the CaV3.3 member of LVA channels by performing a mutational analysis of the two conserved glycines (Gly385 and Gly814), and a valine (Val1383) in IIIS6 which, according to sequence similarity, corresponds to a hinge glycine in KV and NaV channels. Substitution of Gly814 for proline (G814P) or alanine (G814A), as well as the mutant V1383G, drastically decreased whole-cell current density, with discrete effects on the voltage-dependence of activation. However, the mutant G814P displayed a 10 mV shift in the steady-state inactivation curve to hyperpolarized potentials; the current inactivation kinetics was slowed down by 50%, and the recovery was also more than two-fold slower. The substitution by proline also increased the channel closing time constant by 60%; this effect was also displayed by mutant V1383G, which in addition induced the strongest effect on the inactivation kinetics slowing (≥ 2-fold). Our preliminary results suggest that residues Gly814 and Val1383 are mostly involved in the inactivation gating and, in a lesser degree, stabilizing the open state of CaV3.3. Additional mutations, including double and triple substitutions, are currently under investigation.Supported by CONACYT Mexico J50250Q.

Chimera of CaV1.2 and CaV3.1 alpha1 Subunits Suggests Role of the C-terminal Tail in Cytosolic Mg2+ Actions on CaV1.2 Gating

Previous studies (Wang and Berlin, Am. J. Physiol. 291:C83, 2006) have shown that gating properties of CaV1.2 channels (subunits α1, β2A and α2δ) expressed in tsA201 cells are significantly altered by varying cytosolic Mg2+ across a range of physiologic concentrations. Alterations in gating include changes in peak current amplitude as well as kinetics of current inactivation. In contrast, when CaV3.1 (α1 subunit only) is expressed, varying cytosolic Mg2+ across a similar concentration range has little or no effect on channel gating. To understand the molecular basis for the effects of cytosolic Mg2+ on these related Ca2+ channels, a chimera channel consisting of CaV3.1 (α1 residues 1-1826) with the C-terminal region of CaV1.2 (α1 residues 1515-2171) was constructed and expressed in tsA201 cells. Ca2+ currents were measured in cells whole-cell patch-clamped with electrodes containing salt solutions in which Mg2+ and Ca2+ concentrations were strongly buffered. The chimera Ca2+ channel had a similar membrane potential dependence for activation and steady-state inactivation as CaV3.1; however, the rate of current inactivation was slowed at least two-fold. Varying patch electrode Mg2+ concentration had little effect on the rate of current inactivation, similar to CaV3.1, but unlike CaV1.2. On the other hand, current amplitude was depressed in the chimera channel with increasing Mg2+. These results show that the C-terminal tail of CaV1.2 affects kinetics of channel gating. At least in part, changes in channel availability with cytosolic Mg2+ can be attributed to the C-terminal tail of CaV1.2; however, this domain alone cannot be responsible for Mg2+-dependent regulation of channel gating kinetics.

Co-expression of KCNE2 and KChIP2c modulates the electrophysiologi-cal properties of Kv4.2 current in COS-7 cells

Several beta-subunits have been suggested to modulate the electrophysiological properties of the transient outward current (I(to)) in cardiac myocytes, including the obligatory beta-subunit K+-channel interacting protein (KChIP2) and KCNE2. However, neither KChIP2 nor KCNE2 modulation of Kv4.x (x=2 and/or 3) can fully recapitulate the electrophysiological properties of native I(to). The present study is to investigate how I(to) current is modulated when both KChIP2 and KCNE2 are coexpressed. Kv4.2, KChIP2c, and KCNE2 cDNA were simultaneously transfected into COS-7 cells at a molar ratio of 3:1:1. Whole-cell currents were recorded by the patch-clamp method. In comparison with the current regulated by KChIP2c alone, the co-expression of KCNE2 further slowed Kv4.2 current inactivation kinetics, but diminished KChIP2c-induced positive shift of the voltage-dependent activation of Kv4.2 current. Importantly, co-expression of KCNE2 accelerated the current recovery from inactivation, and caused an povershootq of peak current amplitude during Kv4.2 current recovery, a phenomenon which has been uniquely described for human I(to). However, co-expression of KCNE2 exerted no further effect on Kv4.2 current amplitude, the rate of Kv4.2 current activation and voltage-dependent inactivation. Co-expression of Kv4.2 with KChIP2c and KCNE2, but not with KChIP2c or KCNE2 alone, yields a current profile similar to native I(to). Both KChIP2c and KCNE2 simultaneously participate in recapitulation of the electrophysiological properties of I(to) in cardiac myocytes.

Hypoxia Inhibits the Changes in Action Potentials and Ion Channels during Primary Culture of Neonatal Rat Ventricular Myocytes

Action potentials of rat ventricular myocytes are progressively shortened after birth within several weeks mainly due to a progressive increase in transient outward potassium current (Ito). On the supposition that an elevation in blood oxygen after birth may contribute to such developmental change, we studied effects of long-term exposure to hypoxia on changes in cardiac action potentials and Ito. Single ventricular myocytes isolated from day-old neonatal rat hearts were cultured in normoxic condition (21% O2) for 15 days and served as control. To test the influence of long-term exposure to hypoxia, O2tension was reduced to 7.5% at day 6 during culture. In 15-day cells cultured in normoxia, action potential duration (APD) was shortened by 44% (n=11) compared with 5-day cells (n=10); cell capacitance was increased to 2.0-fold. Itodensity was increased by 189–265% (n=11) at voltage levels from −20 to 50 mV without any changes in the kinetics of current inactivation. In 15-day cells cultured in hypoxia, APD was shortened only by 16% (n=6) from control; the increment of cell capacitance was 2.1-fold (n=6). The Itoincrement was limited to 53% (n=8); both inactivation and its recovery of the current was apparently slowed due to the amplification of the slower component. These results suggest that the developmental augmentation of Itoexpression during culture requires oxygen and the increase in Itoand cell hypertrophy are likely regulated independently.

Molecular Diversity of the Calcium Channel α2δ Subunit

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.

Sodium Channel-Blocking Properties of Spiradoline, a κ Receptor Agonist, are Responsible for Its Antiarrhythmic Action in the Rat

Spiradoline (U-62,066E), a selective kappa (kappa) receptor agonist, was examined for actions on the cardiovascular system and on myocardial ionic currents in rats. We initially characterized cardiac, hemodynamic, and antiarrhythmic actions of spiradoline in isolated perfused rat hearts and pentobarbital-anesthetized rats. Electrophysiologic studies in isolated myocytes were used to elucidate the mechanism for changes observed in vivo in the ECG, as well as for antiarrhythmic actions against electrical and ischemia-induced arrhythmias. In isolated rat hearts, spiradoline reduced heart rate and cardiac contractility and increased the PR interval and QRS width of the ECG in a concentration-dependent manner. In anesthetized rats, spiradoline dose-dependently reduced blood pressure and heart rate and prolonged the PR interval and QRS width. At slightly higher doses, it increased the QaT interval of the ECG. RSh, an index of sodium channel blockade in the rat, also was dose-dependently increased. Electrical stimulation of the left ventricle suggested that spiradoline may exert its antiarrhythmic action by blockade of myocardial sodium currents. The electrophysiologic actions of spiradoline on sodium currents, the transient outward (i(to)) and sustained plateau potassium (ik(sus)) currents were studied in isolated cardiac rat myocytes by whole-cell patch-clamp techniques. Spiradoline (15-500 microM) reduced peak sodium current in a rapid, reversible, and concentration-dependent manner; it also increased the rate of decay of I(to) and reduced the amplitude of Ik(sus). At a concentration of 150 microM, spiradoline produced a 24 +/- 2 mV hyperpolarizing shift in sodium current inactivation kinetics but did not alter activation processes. Spiradoline showed both tonic and frequency-dependent components of sodium current block. Thus spiradoline produced its antiarrhythmic actions via sodium channel blockade in myocardial tissue, although higher doses also block potassium currents. This combined ion channel-blocking property may be of added clinical benefit in the setting of myocardial ischemia.

Voltage-Gated K+Channel β Subunits: Expression and Distribution of Kvβ1 and Kvβ2 in Adult Rat Brain

Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.

μ-Opioid-receptor-mediated inhibidon of the N-type calcium-channel current

The predominant consequences of mu-opioid-receptor activation are depression of both neuronal activity and transmitter release. Mu-Opioid agonists have previously been observed to increase a potassium conductance and to inhibit adenylate cyclase. We now report that activation of mu-opioid receptors directly decreases the N-type calcium-channel current in a differentiated, human neuroblastoma cell line (SH-SY5Y). The coupling between the mu-opioid receptor and the calcium channel involves a pertussis toxin-sensitive G protein and is independent of changes in adenylate cyclase activity. The inhibition of the calcium-channel current is voltage dependent because it is largely overcome by strong membrane depolarization. It is not associated with changes in the kinetics of current inactivation. Therefore, the mu-receptor belongs to the superfamily of G-protein-coupled, inhibitory neurotransmitter receptors which modulate the activity of calcium and potassium channels and adenylate cyclase.