Persistent Na Currents Research Articles

Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a "gain-of-function" defect, and Brugada syndrome, a "loss-of-function" phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

P1596Interaction of CaMKII and NaV1.8 modulates cardiac electrophysiology in human heart failure

Abstract Introduction In human heart failure, electrical remodeling contributes to the risk of arrhythmia generation. Increased expression of Ca/Calmodulin-dependent protein kinase IIδ (CaMKIIδ) and an enhanced persistent Na current (INaL) have been linked to arrhythmogenesis. CaMKIIδ increases INaL via regulation of sodium channels thereby contributing to arrhythmias through early- and delayed-afterdepolarizations (EADs and DADs). Genome-wide association studies (GWAS) have described the implication of the neuronal sodium channel isoform NaV1.8 (SCN10A) in cardiac electrophysiology showing modulation in cardiac conduction. We showed that the expression of the isoform Nav1.8 is significantly increased in human failing cardiomyocytes and contributes substantially to the enhanced INaL. Purpose We investigated a potential interaction of CaMKIIδ and NaV1.8 and thereby its role in arrhythmia generation and electrophysiology in human and murine failing hearts. Methods Cardiomyocytes were isolated from explanted failing hearts and CaMKIIδ transgenic (TG) mice. We performed immunostainings and co-immunoprecipitation (Co-IP) to show interactions of CaMKIIδ and Nav1.8 in isolated cardiomyocytes and homogenates. Whole-cell patch clamp experiments were conducted in isolated human and murine ventricular cardiomyocytes. Additionally, Ca2+ transients were measured using epifluorescence microscopy with the Ca2+ dye fura-2 (10μmol/L) whereas Ca2+ sparks measurements were performed by using confocal microscopy with the Ca2+ dye fluo-4 (10μmol/L). PF-01247324 is a novel specific NaV1.8 inhibitor (orally bioavailable; 1 μmol/L) and autocamtide inhibitory peptide (AIP, 1 μmol/L) was used to inhibit CaMKIIδ. Results Co-immunoprecipitation experiments revealed an association of CaMKIIδ and Nav1.8 in human homogenates compared to healthy controls. Furthermore, immunohistochemistry stainings in isolated human cardiomyocytes showed a co-localization of CaMKIIδ and NaV1.8 at the intercalated disc and t-tubules. We observed a significant reduction of INaL integral and proarrhythmic SR-Ca2+ spark frequency (CaSpF) after addition of either PF-01247324 or the CaMKIIδ inhibitor AIP in failing human and murine ventricular cardiomyocytes. When PF-01247324 and AIP were added together, the decrease in INaL integral and CaSpF was comparable to PF-01247324 alone in human failing cardiomyocytes. Inhibition of NaV1.8 did not show an effect on Ca2+ transient amplitude or Ca2+ transient decay at different stimulation frequencies in CaMKIIδ TG cardiomyocytes. Conclusion Our results demonstrate the significance of both CaMKIIδ and NaV1.8 in INaL generation and their detrimental interaction. This data suggest that increased CaMKIIδ activity plays a substantial role for the activation of NaV1.8-mediated late sodium current and SR-Ca2+ leak.

Cell-attached single-channel recordings in intact prefrontal cortex pyramidal neurons reveal compartmentalized D1/D5 receptor modulation of the persistent sodium current.

The persistent Na+ current (INap) is believed to be an important target of dopamine modulation in prefrontal cortex (PFC) neurons. While past studies have tested the effects of dopamine on INap, the results have been contradictory largely because of difficulties in measuring INap using somatic whole-cell recordings. To circumvent these confounds we used the cell-attached patch-clamp technique to record single Na+ channels from the soma, proximal dendrite (PD) or proximal axon (PA) of intact prefrontal layer V pyramidal neurons. Under baseline conditions, numerous well resolved Na+ channel openings were recorded that exhibited an extrapolated reversal potential of 73 mV, a slope conductance of 14–19 pS and were blocked by tetrodotoxin (TTX). While similar in most respects, the propensity to exhibit prolonged bursts lasting >40 ms was many fold greater in the axon than the soma or dendrite. Bath application of the D1/D5 receptor agonist SKF81297 shifted the ensemble current activation curve leftward and increased the number of late events recorded from the PD but not the soma or PA. However, the greatest effect was on prolonged bursting where the D1/D5 receptor agonist increased their occurrence 3 fold in the PD and nearly 7 fold in the soma, but not at all in the PA. As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region. Therefore, D1/D5 receptor modulation appears to be targeted mainly to Na+ channels in the PD/soma and not the PA. By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of INap, we demonstrate conclusively that D1/D5 receptor activation can increase the INap generated proximally, however questions still remain as to how D1/D5 receptor modulates Na+ currents in the more distal initial segment where most of the INap is normally generated.

Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra.

GABA projection neurons (GABA neurons) in the substantia nigra pars reticulata (SNr) and dopamine projection neurons (DA neurons) in substantia nigra pars compacta (SNc) have strikingly different firing properties. SNc DA neurons fire low-frequency, long-duration spikes, whereas SNr GABA neurons fire high-frequency, short-duration spikes. Since voltage-activated sodium (Na(V)) channels are critical to spike generation, the different firing properties raise the possibility that, compared with DA neurons, Na(V) channels in SNr GABA neurons have higher density, faster kinetics, and less cumulative inactivation. Our quantitative RT-PCR analysis on immunohistochemically identified nigral neurons indicated that mRNAs for pore-forming Na(V)1.1 and Na(V)1.6 subunits and regulatory Na(V)β1 and Na(v)β4 subunits are more abundant in SNr GABA neurons than SNc DA neurons. These α-subunits and β-subunits are key subunits for forming Na(V) channels conducting the transient Na(V) current (I(NaT)), persistent Na current (I(NaP)), and resurgent Na current (I(NaR)). Nucleated patch-clamp recordings showed that I(NaT) had a higher density, a steeper voltage-dependent activation, and a faster deactivation in SNr GABA neurons than in SNc DA neurons. I(NaT) also recovered more quickly from inactivation and had less cumulative inactivation in SNr GABA neurons than in SNc DA neurons. Furthermore, compared with nigral DA neurons, SNr GABA neurons had a larger I(NaR) and I(NaP). Blockade of I(NaP) induced a larger hyperpolarization in SNr GABA neurons than in SNc DA neurons. Taken together, these results indicate that Na(V) channels expressed in fast-spiking SNr GABA neurons and slow-spiking SNc DA neurons are tailored to support their different spiking capabilities.

Persistent Na-channels: Origin and function

Voltage-dependent sodium channels have a decisive role in the generation of action potentials (AP) in many types of cells. In addition to the fast inactivating Na-current, associated with AP generation, the Na-channel can give rise to a noninactivating or persistent Na-current. The latter current generally comprises up to 5% of the transient current having important physiological consequences. It was established that persistent Na-currents have functional significance in setting the membrane potential in a subthreshold range regulating by this way dendritic depolarisations, repetitive firing and enhancing synaptic transmission. Voltage dependent sodium channel genes have been identified in a variety of invertebrates, as well as mammalian and nonmammalian vertebrates. It has been established that the biophysical properties, pharmacology and gene organization of invertebrate sodium channels are largely similar to the vertebrate ones, supporting the view that the ancestral sodium channel was established before the evolutionary separation of the invertebrates from the vertebrates. Although different isoforms of voltage sensitive Na-channels have now been identified the mechanism for persistent current remains controversial. An important yet unanswered question is whether persistent and fast inactivating Na-currents arise from different sets of sodium channels or whether the persistent Na-current results from different gating of the same channel type. The aim of the present review is to discuss the origin and the function of the persistent current, focusing on data derived from an invertebrate animal.

Distribution of calcium binding proteins, sodium channels and persistent sodium current in the rat ventral respiratory group

The ventral respiratory group (VRG) is important in generating both the breathing rhythm and motor pattern. The VRG is generally subdivided into 4 regions, the Botzinger complex, preBotzinger Complex (pBc), rostral VRG, and caudal VRG based on neuron discharge patterns and axonal projections. Anatomical markers provide a potential bridge between the physiological analysis of the respiratory system and the neurochemical elements that are the ultimate building blocks of this system. An example of this approach is the coincidence of NK1 receptors with neurons in the pBc complex [1]. VRG labeling by antibodies to calcium binding proteins (parvalbumin, calbindin, and calretinin) in some respects complements the NK1 immunoreactivity. Parvalbumin has been suggested to label VRG neurons [2]. We have verified this by demonstrating that parvalbumin positive neurons project to the ipsilateral and contralateral VRG as well as to the phrenic nucleus. We have also found that a prominent gap in the column of VRG related parvalbumin cells [2] likely corresponds to the pBc since parvalbumin cells are rare in this zone and never co-localize with NK1 receptors. Calbindin and calretinin immunoreactive cells are scattered in the pBc and rostral VRG but rare in the Botzinger complex. Calbindin neurons are intermingled, but not colocalized with pBc NK1 cells. Calretinin is not colocalized with NK1, except for a small population of cells at the caudal ventral edge of the pBc, which likely corresponds to NK1 bulbospinal neurons [3]. Finally, preliminary evidence indicates glycine immunoreactivity in some parvalbumin neurons within the Botzinger complex region. In addition to this compartmentalized distribution of calcium binding proteins within the VRG, preliminary evidence is consistent with a differential distribution of Na channel alpha subunits. A slowly inactivating persistent Na current is postulated to underlie the pacemaker activity seen in a subset of pBc neurons [4]. Within the CNS at least 5 different Na channel alpha subunits have been identified, termed Nav1.1, 1.2, 1.3, 1.5 and 1.6. Of these Nav1.6 has been most strongly linked to persistent Na current. Immunohistochemical examination of Nav1.2, 1.3 and 1.6 demonstrated Nav1.2 is widely expressed in the VRG including some NK1 neurons. Nav1.3 was present in cranial motoneurons. In neurons acutely dissociated from the pBc of 1–15 day old rats, whole-cell voltage clamp recordings were used to analyze transient and persistent Na currents and single-cell RT-PCR was used to probe for Nav1.1, 1.2 and 1.6 mRNA. Whole-cell recordings were made using an external solution containing 50 mM NaCl. Slow ramp depolarization from -80 to +30 mV revealed a TTX-sensitive, persistent Na current. The current kinetics were dependent upon the ramp speed. A slow ramp (100 mV/s), elicited an inward non-inactivating current in 42% (10 of 24) of neurons sampled from the pBc. These results are consistent with a role for persistent Na current in regulation of the subthreshold behavior, including pacemaker activity. Moreover, single-cell RT-PCR revealed the presence of Nav1.6 in 40 of 72 cells (55%). The Nav1.1 subunit mRNA was present in 47 of 82 neurons (57%) and was co-expressed with Nav1.6 in 28% of cells. Preliminary findings on Nav1.2 mRNA are consistent with the immunohistochemistry with it present in 7 of 18 (38%) pBc neurons.

Sodium channel inactivation is impaired in equine hyperkalemic periodic paralysis.

1. Equine hyperkalemic periodic paralysis (E-HPP) is a dominantly inherited disorder of muscle that causes recurrent episodes of stiffness (myotonia) and weakness in association with elevated serum K+. Affected horses carry a mutant allele of the skeletal muscle isoform of the Na channel alpha-subunit. To understand how this mutation may cause the disease phenotype, the functional defect in Na channel behavior was defined physiologically by recording unitary currents from cell-attached patches on normal and affected equine myotubes. 2. The presence of the mutation was confirmed in our cell line by restriction digest of polymerase chain reaction (PCR)-amplified genomic DNA. Myotubes from the affected horse were heterozygous for the point mutation that codes for a Phe to Leu substitution in S3 of domain IV. This assay provides a rapid technique to screen for the mutation in horses at risk. 3. The primary physiological defect in mutant Na channels was an impairment of inactivation. This defect was manifest as bursts of persistent activity during which the channel closed and reopened throughout a maintained depolarization. Disrupted inactivation slowed the decay of the ensemble-averaged current and produced an eightfold increase in the steady-state open probability measured at the end of a 40-ms pulse. This point mutation identifies a new region of the alpha subunit that is important for rapid inactivation of the channel. 4. The persistent Na current was produced by a distinct mode of gating. Failure of a mutant channel to inactivate was infrequent and occurred in groups of consecutive trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of diazoxide on CA1 neurons in hippoeampal slices from rats

Membrane effects of diazoxide (DZX) were examined in CA1 pyramidal neurons, mainly by whole-cell recording in slices kept at 33 degrees C (from Sprague-Dawley rats). Bath applications of DZX (0.65 mM) did not significantly change the resting input conductance; but instantaneous inward rectification was reduced by 47 +/- 14% (near -110 mV). There was a similar depression of a large, sustained voltage-dependent outward current (by 44 +/- 11% near 0 mV). A nearly identical reduction of the outward current recorded in a Ca current suppressing medium (but not in 30 mM tetraethylammonium) indicated that the DZX-sensitive current includes the delayed rectifier. In Mn, low-Ca medium containing tetraethylammonium and carbachol, DZX potentiated (by 43 +/- 12%) the D-type slowly decaying outward current seen after hyperpolarizing pulses at a holding potential of approximately -50 mV. DZX abolished or depressed slow inward currents, such as the tetrodotoxin-sensitive persistent Na current, high voltage activated Ca currents (IC50 = 0.47 mM), and the Q current. In 6 of 13 cells recorded with electrodes containing either guanosine or adenosine diphosphate, DZX potentiated the voltage-dependent outward current, but input conductances were reduced. In conclusion, although there was little indication that it activates classical KATP channels in CA1 neurons, DZX strongly depresses several voltage-dependent, slowly inactivating outward and inward currents, which are important modulators of cell excitability.

A persistent, TTX-sensitive sodium current in an invertebrate neuron with neurosecretory ultrastructure

In the CNS of the giant barnacle (Balanus nubilus) a single pair of large neuronal somata (cross-commissural, or CC, cells), located near the entry of the median ocellar nerve, occasionally displays a prominent whitish luster. These somata have ultrastructure typical of neurosecretory cells: numerous Golgi complexes and abundant, large dense-cored vesicles (DCVs; size range, 75-275 nm). Injection of a CC cell with cobalt tracer shows that it arborizes over a 7 mm length of the contralateral peripheral nerve out of which it projects. The processes of the arbor are profuse and varicose; the varicosities are packed with DCVs similar to those in the soma. Stimulation of a single CC cell causes a substantial decrease in the number of DCVs and increases the incidence of clusters of small electron-lucent vesicles, as well as the occurrence of large electron-lucent vesicles and membrane-bound cisternae. We studied ionic currents flowing across this cell's somatic membrane with a single-electrode voltage clamp. Unusual among these currents is an inward current that is blocked by TTX but is essentially noninactivating. In current clamp, this "persistent" current causes the action potential to be prolonged (seconds) if opposing outward current is blocked with 4-aminopyridine. The inward current is carried by Na. Its amplitude depends on the external Na concentration, it is blockable by TTX, and it persists when the cell is bathed in Ca-free saline and/or Co. Other currents present in this cell include an outward current similar to molluscan A-current and a Ca current that contributes to the action potential (Stockbridge and Ross, 1986). The persistent Na current is partially activated at the cell's resting potential and, thus, may participate in determining the frequency of its impulse activity.