Shift Of Steady-state Inactivation Research Articles

Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome.

Duplication of the maternal chromosome 15q11.2-q13.1 region causes Dup15q syndrome, a highly penetrant neurodevelopmental disorder characterized by severe autism and refractory seizures. Although UBE3A, the gene encoding the ubiquitin ligase E3A, is thought to be the main driver of disease phenotypes, the cellular and molecular mechanisms that contribute to the development of the syndrome are yet to be determined. We previously established the necessity of UBE3A overexpression for the development of cellular phenotypes in human Dup15q neurons, including increased action potential firing and increased inward current density, which prompted us to further investigate sodium channel kinetics. We used a Dup15q patient-derived induced pluripotent stem cell line that was CRISPR-edited to remove the supernumerary chromosome and create an isogenic control line. We performed whole cell patch clamp electrophysiology on Dup15q and corrected control neurons at two time points of in vitro development. Compared to corrected neurons, Dup15q neurons showed increased sodium current density and a depolarizing shift in steady-state inactivation. Moreover, onset of slow inactivation was delayed, and a faster recovery from both fast and slow inactivation processes was observed in Dup15q neurons. A fraction of sodium current in Dup15q neurons (~15%) appeared to be resistant to slow inactivation. Not unexpectedly, a higher fraction of persistent sodium current was also observed in Dup15q neurons. These phenotypes were modulated by the anticonvulsant drug rufinamide. Sodium channels play a crucial role in the generation of action potentials, and sodium channelopathies have been uncovered in multiple forms of epilepsy. For the first time, our work identifies in Dup15q neurons dysfunctional inactivation kinetics, which have been previously linked to multiple forms of epilepsy. Our work can also guide therapeutic approaches to epileptic seizures in Dup15q patients and emphasize the role of drugs that modulate inactivation kinetics, such as rufinamide.

SCN1A gain-of-function mutation causing an early onset epileptic encephalopathy.

Loss-of-function variants in SCN1A cause Dravet syndrome, the most common genetic developmental and epileptic encephalopathy (DEE). However, emerging evidence suggests separate entities of SCN1A-related disorders due to gain-of-function variants. Here, we aim to refine the clinical, genetic, and functional electrophysiological features of a recurrent p.R1636Q gain-of-function variant, identified in four individuals at a single center. Individuals carrying the recurrent SCN1A p.R1636Q variant were identified through diagnostic testing. Whole cell voltage-clamp electrophysiological recording in HEK-293 T cells was performed to compare the properties of sodium channels containing wild-type Nav 1.1 or Nav 1.1-R1636Q along with both Nav β1 and Nav β2 subunits, including response to oxcarbazepine. To delineate differences from other SCN1A-related epilepsies, we analyzed electronic medical records. All four individuals had an early onset DEE characterized by focal tonic seizures and additional seizure types starting in the first few weeks of life. Electrophysiological analysis showed a mixed gain-of-function effect with normal current density, a leftward (hyperpolarized) shift of steady-state inactivation, and slower inactivation kinetics leading to a prominent late sodium current. The observed functional changes closely paralleled effects of pathogenic variants in SCN3A and SCN8A at corresponding positions. Both wild type and variant exhibited sensitivity to block by oxcarbazepine, partially correcting electrophysiological abnormalities of the SCN1A p.R1636Q variant. Clinically, a single individual responded to treatment with oxcarbazepine. Across 51 individuals with SCN1A-related epilepsies, those with the recurrent p.R1636Q variants had the earliest ages at onset. The recurrent SCN1A p.R1636Q variant causes a clinical entity with a wider clinical spectrum than previously reported, characterized by neonatal onset epilepsy and absence of prominent movement disorder. Functional consequences of this variant lead to mixed loss and gain of function that is partially corrected by oxcarbazepine. The recurrent p.R1636Q variant represents one of the most common causes of early onset SCN1A-related epilepsies with separate treatment and prognosis implications.

Novel G1481V and Q1491H SCN5A Mutations Linked to Long QT Syndrome Destabilize the Nav1.5 Inactivation State

BackgroundNav1.5, which is encoded by the SCN5A gene, is the predominant voltage-gated Na+ channel in the heart. Several mutations of this gene have been identified and reported to be involved in several cardiac rhythm disorders, including type 3 long QT interval syndrome, that can cause sudden cardiac death. We analyzed the biophysical properties of 2 novel variants of the Nav1.5 channel (Q1491H and G1481V) detected in 5- and 12-week-old infants diagnosed with a prolonged QT interval. MethodsThe Nav1.5 wild-type and the Q1491H and G1481V mutant channels were reproduced in vitro. Wild-type or mutant channels were cotransfected in human embryonic kidney (HEK) 293 cells with the beta 1 regulatory subunit. Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. ResultsThe Q1491H mutant channel exhibited a lower current density, a persistent Na+ current, an enhanced window current due to a +20-mV shift of steady-state inactivation, a +10-mV shift of steady-state activation, a faster onset of slow inactivation, and a recovery from fast inactivation with fast and slow time constants of recovery. The G1481V mutant channel exhibited an increase in current density and a +7-mV shift of steady-state inactivation. The observed defects are characteristic of gain-of-function mutations typical of type 3 long QT interval syndrome. ConclusionsThe 5- and 12-week-old infants displayed prolonged QT intervals. Our analyses of the Q1491H and G1481V mutations correlated with the clinical diagnosis. The observed biophysical dysfunctions associated with both mutations were most likely responsible for the sudden deaths of the 2 infants.

Biophysical defects of an SCN5A V1667I mutation associated with epinephrine-induced marked QT prolongation.

The epinephrine infusion test (EIT) typically induces marked QT prolongation in LQT1, but not LQT3, while the efficacy of β-blocker therapy is established in LQT1, but not LQT3. We encountered an LQT3 family, with an SCN5A V1667I mutation, that exhibited epinephrine-induced marked QT prolongation. Wild-type (WT) or V1667I-SCN5A was transiently expressed into tsA-201 cells, and whole-cell sodium currents (INa ) were recorded using patch-clamp techniques. To mimic the effects of epinephrine, INa was recorded after the application of protein kinase A (PKA) activator, 8-CPT-cAMP (200 μM), for 10 minutes. The peak density of V1667I-INa was significantly larger than WT-INa (WT: 469 ± 48 pA/pF, n = 20; V1667I: 690 ± 62 pA/pF, n = 19, P < .01). The steady-state activation (SSA) and fast inactivation rate of V1667I-INa were comparable to WT-INa . V1667I-INa displayed a significant depolarizing shift in steady-state inactivation (SSI) in comparison to WT-INa (V1/2 -WT: -88.1 ± 0.8 mV, n = 17; V1667I: -82.5 ± 1.1 mV, n = 17, P < .01), which increases window currents. Tetrodotoxin (30 μM)-sensitive persistent V1667I-INa was comparable to WT-INa . However, the ramp pulse protocol (RPP) displayed an increased hump in V1667I-INa in comparison to WT-INa . Although 8-CPT-cAMP shifted SSA to hyperpolarizing potentials in WT-INa and V1667I-INa to the same extent, it shifted SSI to hyperpolarizing potentials much less in V1667I-INa than in WT-INa (V1/2 -WT: -92.7 ± 1.3 mV, n = 6; V1667I: -85.3 ± 1.6 mV, n = 6, P < .01). Concordantly, the RPP displayed an increased hump in V1667I-INa , but not in WT-INa . We demonstrated an increase of V1667I-INa by PKA activation, which may provide a rationale for the efficacy of β-blocker therapy in some cases of LQT3.

Revisiting NaV Channel Inactivation: The Role of Fibroblast Growth Factor Homologous Factor (FHF)

Cardiac sodium channel (NaV1.5) activation underlies the initiation and propagation of the heartbeat and is regulated by numerous accessory proteins. NaV1.5 comprises 4 homologous domains (DI-DIV), each consisting of 6 transmembrane segments (S1-S6) constituting the voltage sensing domain (VSD; S1-S4) and the channel pore (S5-S6). FHF, or intracellular fibroblast growth factors (iFGF), were found to regulate NaV1.5 kinetics through C-terminal binding near Calmodulin (CaM), but the mechanism remains unclear. To probe this mechanism, we observed the regulation of Nav1.5 VSDs by the cardiac-predominant FHF1B (iFGF12B), and the alternative FHF1A (iFGF12A) splice variant, using voltage-clamp fluorometry (VCF) in the cut-open configuration for individual VSD monitoring. Voltage clamp and patch clamp were also employed for electrophysiological studies in Xenopus Oocytes and CHO cells. We found that FHF1A and FHF1B regulate NaV1.5 inactivation by causing depolarizing shifts in the steady-state inactivation (SSI) curve and the DIII-VSD activation. Disruption of the CaM binding motif (IQ/AA) did not alter the SSI shift, suggesting CaM-independent FHF modulation. However, mutation of the inactivation gate (IFM motif) within DIII-IV linker (F1486Q) negates the FHF's effects on DIII-VSD and SSI. We conclude that FHF1s regulate NaV1.5 inactivation through an inactivation gate, independent of CaM. Furthermore, the A-type FHF counteracts impaired inactivation of F1486Q-NaV1.5 and reduces the late sodium current. A peptide bearing the first 21 amino acids of FHF1A's N-terminus is insufficient to reduce the late sodium current, implying different mechanism than pore blocking. Similar inactivation rescue by FHF1A was observed in long QT syndrome-linked mutations (ΔKPQ, ΔK1500 and F1473S), without affecting the shifts in SSI nor DIII-VSD. Results suggest possible FHF1A function as natural antiarrhythmic agent, that partially restore the inactivation impaired by mutations through an inactivation gate stabilization, independent of its connection to DIII-VSD.

"Pill-in-the-Pocket" Treatment of Propafenone Unmasks ECG Brugada Pattern in an Atrial Fibrillation Patient With a Common SCN5A R1193Q Polymorphism.

Background: “Pill-in-the-pocket” (PIP) treatment with type IC drugs for cardioversion of recent-onset atrial fibrillation (AF) has been recommended in guidelines. Major adverse effects have been often reported, and the underlying mechanisms are proposed to be associated with the genetic backgrounds.Methods and Results: A male patient was treated with PIP approach (propafenone 600 mg.po) for the conversion of new onset AF. His symptoms got worse and referred to emergency room; ECG showed a typical Brugada syndrome (BrS) type I ECG pattern with sinus rhythm. Genetic screening identified a common SCN5A polymorphism R1193Q. Propafenone blockade of INa was studied in HEK293 cells expressed SCN5A R1193Q channel and WT channel using patch clamp techniques. There was no significant difference in peak current and steady-state gating parameters between R1193Q and WT at baseline. At clinically relevant concentration of 2 μmol/L propafenone, use-dependent block (UDB) of INa was more pronounced in R1193Q versus WT (44.2 ± 7.2 versus 24.8 ± 5.7% at the frequency of 2 Hz, P < 0.05); IC50 of UDB was 2.9 ± 0.7 μmol/L for R1193Q and 8.1 ± 1.8 μmol/L for WT, respectively. Propafenone produced more left shift of steady-state inactivation and slower recovery from inactivation in R1193Q compared with WT.Conclusion: A common SCN5A polymorphism R1193Q enhances UDB by propafenone and predisposes the patients to drug-induced BrS with PIP treatment. Our data suggest that R1193Q polymorphism is likely to be a genetic marker for the major adverse effects associated with propafenone PIP approach for AF patients’ management. Ajmaline challenge to rule out the presence of BrS should be considered prior to propafenone PIP therapy in AF patients who are identified to have R1193Q polymorphism.

Conditional knockout of Fgf13 in murine hearts increases arrhythmia susceptibility and reveals novel ion channel modulatory roles

The intracellular fibroblast growth factors (iFGF/FHFs) bind directly to cardiac voltage gated Na+ channels, and modulate their function. Mutations that affect iFGF/FHF-Na+ channel interaction are associated with arrhythmia syndromes. Although suspected to modulate other ionic currents, such as Ca2+ channels based on acute knockdown experiments in isolated cardiomyocytes, the in vivo consequences of iFGF/FHF gene ablation on cardiac electrical activity are still unknown. We generated inducible, cardiomyocyte-restricted Fgf13 knockout mice to determine the resultant effects of Fgf13 gene ablation. Patch clamp recordings from ventricular myocytes isolated from Fgf13 knockout mice showed a ~25% reduction in peak Na+ channel current density and a hyperpolarizing shift in steady-state inactivation. Electrocardiograms on Fgf13 knockout mice showed a prolonged QRS duration. The Na+ channel blocker flecainide further prolonged QRS duration and triggered ventricular tachyarrhythmias only in Fgf13 knockout mice, suggesting that arrhythmia vulnerability resulted, at least in part, from a loss of functioning Na+ channels. Consistent with these effects on Na+ channels, action potentials in Fgf13 knockout mice, compared to Cre control mice, exhibited slower upstrokes and reduced amplitude, but unexpectedly had longer durations. We investigated candidate sources of the prolonged action potential durations in myocytes from Fgf13 knockout mice and found a reduction of the transient outward K+ current (Ito). Fgf13 knockout did not alter whole-cell protein levels of Kv4.2 and Kv4.3, the Ito pore-forming subunits, but did decrease Kv4.2 and Kv4.3 at the sarcolemma. No changes were seen in the sustained outward K+ current or voltage-gated Ca2+ current, other candidate contributors to the increased action potential duration. These results implicate that FGF13 is a critical cardiac Na+ channel modulator and Fgf13 knockout mice have increased arrhythmia susceptibility in the setting of Na+ channel blockade. The unanticipated effect on Ito revealed new FGF13 properties and the unexpected lack of an effect on voltage-gated Ca2+ channels highlight potential compensatory changes in vivo not readily revealed with acute Fgf13 knockdown in cultured cardiomyocytes.

Modulation Effects of Cordycepin on Voltage-Gated Sodium Channels in Rat Hippocampal CA1 Pyramidal Neurons in the Presence/Absence of Oxygen.

Our previous study revealed that cordycepin features important neuroprotective effects against hypoxic insult by improvement of neuronal electrophysiological function. Modulation on voltage-gated sodium channel (VGSC) in CA1 neurons is the initial event during hypoxia/ischemia. However, no study comprehensively investigated cordycepin on VGSC. Hence, this study investigated modulation effects of cordycepin on VGSC not only in oxygen physiological conditions but also in acute oxygen deprivation injury conditions. Results revealed that cordycepin (80 μM) reduced the amplitude of VGSC currents (INa) (77.6% of control, p < 0.01) within 1 min of drug exposure coupled with a negative shift in steady-state inactivation and prolonged recovery time course from inactivation. Additionally, this mild reduction on the peak of INa induced by the pretreatment with cordycepin can attenuate and delay the following hypoxia causing rapid dramatic decrease in INa with no additive change in the voltage dependence of inactivation. As modulation on VGSC in CA1 neurons represents the initial event during ischemia, we propose that suppression effect of cordycepin on VGSC is an important neuronal protective mechanism that may enhance neuronal tolerance to acute oxygen deprivation and delay hypoxia-induced neuronal injuries.

Pathogenic mechanism of recurrent mutations of SCN8A in epileptic encephalopathy.

The early infantile epileptic encephalopathy type 13 (EIEE13, OMIM #614558) results from de novo missense mutations of SCN8A encoding the voltage-gated sodium channel Nav1.6. More than 20% of patients have recurrent mutations in residues Arg1617 or Arg1872. Our goal was to determine the functional effects of these mutations on channel properties. Clinical exome sequencing was carried out on patients with early-onset seizures, developmental delay, and cognitive impairment. Two mutations identified here, p.Arg1872Leu and p.Arg1872Gln, and two previously identified mutations, p.Arg1872Trp and p.Arg1617Gln, were introduced into Nav1.6 cDNA, and effects on electrophysiological properties were characterized in transfected ND7/23 cells. Interactions with FGF14, G-protein subunit Gβγ, and sodium channel subunit β1 were assessed by coimmunoprecipitation. We identified two patients with the novel mutation p.Arg1872Leu and one patient with the recurrent mutation p.Arg1872Gln. The three mutations of Arg1872 and the mutation of Arg1617 all impaired the sodium channel transition from open state to inactivated state, resulting in channel hyperactivity. Other observed abnormalities contributing to elevated channel activity were increased persistent current, increased peak current density, hyperpolarizing shift in voltage dependence of activation, and depolarizing shift in steady-state inactivation. Protein interactions were not affected. Recurrent mutations at Arg1617 and Arg1872 lead to elevated Nav1.6 channel activity by impairing channel inactivation. Channel hyperactivity is the major pathogenic mechanism for gain-of-function mutations of SCN8A. EIEE13 differs mechanistically from Dravet syndrome, which is caused by loss-of-function mutations of SCN1A. This distinction has important consequences for selection of antiepileptic drugs and the development of gene- and mutation-specific treatments.

MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels

The transmembrane S6 segments of Na+ sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na+ channel permeation, gating, and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET) to investigate the role of the S6 segment of homologous domain 4 (D4S6) in the gating of the cardiac (Nav1.5) channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na+ currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C) and C-terminus (Y1767C) of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C) where MTSET increased the persistent Na+ current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

Conservation of Calmodulin Regulation Across Sodium and Calcium Channels

Voltage-gated Na and Ca2+ channels comprise two major ion-channel superfamilies supporting divergent biology and biophysics. Yet, the carboxyl tails of these channels exhibit remarkable homology, hinting at a long-shared and purposeful module. If homologous tail domains elaborated functions of like correspondence, the common origins of such a module could be assured. Indeed, dynamic interactions between calmodulin and tail domains of Ca2+ channels elaborate robust and recognizably similar forms of Ca2+ regulation. However, years of Na channel research have only revealed subtle and variable Ca2+ effects, with divergent mechanisms. These studies employed BAPTA or EGTA to statically elevate intracellular Ca2+ to 1-10 micromolar, a range well above their dissociation constants (A). With Ca2+ appropriately buffered using HEDTA (A), no discernible Ca2+-dependent shift in steady-state inactivation of Na channels was observed (B), thus intensifying the discordance between homology and function and casting doubt on prior structure-function studies. However, using Ca2+ photouncaging, we find that these dissimilarities in Na channels are only apparent. Indeed, Ca2+ regulatory function and mechanism are fundamentally conserved across channel superfamilies, thus substantiating the preservation of an ancient Ca2+-regulatory design featuring calmodulin as a modulator throughout much of living history.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Ion Channel Modulation in Spinal/Trigeminal Synaptic Plasticity

Ion Channel Modulation in Spinal/Trigeminal Synaptic Plasticity

Nociceptive Neurons Differentially Express Fast and Slow T-Type Ca2+Currents in Different Types of Diabetic Neuropathy

T-type Ca2+ channels are known as important participants of nociception and their remodeling contributes to diabetes-induced alterations of pain sensation. In this work we have established that about 30% of rat nonpeptidergic thermal C-type nociceptive (NTCN) neurons of segments L4–L6 express a slow T-type Ca2+ current (T-current) while a fast T-current is expressed in the other 70% of these neurons. Streptozotocin-induced diabetes in young rats resulted in thermal hyperalgesia, hypoalgesia, or normalgesia 5-6 weeks after the induction. Our results show that NTCN neurons obtained from hyperalgesic animals do not express the slow T-current. Meanwhile, the fraction of neurons expressing the slow T-current did not significantly change in the hypo- and normalgesic diabetic groups. Moreover, the peak current density of fast T-current was significantly increased only in the neurons of hyperalgesic group. In contrast, the peak current density of slow T-current was significantly decreased in the hypo- and normalgesic groups. Experimental diabetes also resulted in a depolarizing shift of steady-state inactivation of fast T-current in the hyperalgesic group and slow T-current in the hypo- and normalgesic groups. We suggest that the observed changes may contribute to expression of different types of peripheral diabetic neuropathy occurring during the development of diabetes mellitus.

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNaV1.4F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNaV1.4F1705I are incompletely understood. The location of the hNaV1.4F1705I in the CT prompted us to examine the role of Ca2+ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human NaV1.4 channel regulation by Ca2+ and CaM. hNaV1.4F1705I inactivation gating is Ca2+-sensitive compared to wild type hNaV1.4 which is Ca2+ insensitive and the mutant channel exhibits a depolarizing shift of the V1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNaV1.4 channel background (rNaV1.4F1698I) eliminates Ca2+ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca2+ sensitivity of gating between wild type and mutant human and rat NaV1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca2+/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows INa decay in a Ca2+-sensitive fashion. The combination of the altered voltage dependence and kinetics of INa decay contribute to the myotonic phenotype and may involve the Ca2+-sensing apparatus in the CT of NaV1.4.

Modeling type 3 long QT syndrome with cardiomyocytes derived from patient-specific induced pluripotent stem cells

BackgroundType 3 long QT syndrome (LQT3) is the third most common form of LQT syndrome and is characterized by QT-interval prolongation resulting from a gain-of-function mutation in SCN5A. We aimed to establish a patient-specific human induced pluripotent stem cell (hiPSC) model of LQT3, which could be used for future drug testing and development of novel treatments for this inherited disorder. Methods and resultsDermal fibroblasts obtained from a patient with LQT3 harboring a SCN5A mutation (c.5287G>A; p.V1763M) were reprogrammed to hiPSCs via repeated transfection of mRNA encoding OCT-4, SOX-2, KLF-4, C-MYC and LIN-28. hiPSC-derived cardiomyocytes (hiPSC-CMs) were obtained via cardiac differentiation. hiPSC-CMs derived from the patient's healthy sister were used as a control. Compared to the control, patient hiPSC-CMs exhibited dominant mutant SCN5A allele gene expression, significantly prolonged action potential duration or APD (paced CMs of control vs. patient: 226.50±17.89ms vs. 536.59±37.1ms; mean±SEM, p<0.005), an increased tetrodotoxin (TTX)-sensitive late or persistent Na+ current (control vs. patient: 0.65±0.11 vs. 3.16±0.27pA/pF; n=9, p<0.01), a positive shift of steady state inactivation and a faster recovery from inactivation. Mexiletine, a NaV1.5 blocker, reversed the elevated late Na+ current and prolonged APD in LQT3 hiPSC-CMs. ConclusionsWe demonstrate that hiPSC-CMs derived from a LQT3 patient recapitulate the biophysical abnormalities that define LQT3. The clinical significance of such an in vitro model is in the development of novel therapeutic strategies and a more personalized approach in testing drugs on patients with LQT3.

The Auxiliary Subunit KChIP2 Is an Essential Regulator of Homeostatic Excitability

The necessity for, or redundancy of, distinctive KChIP proteins is not known. Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. KChIP2 is essential for homeostasis in hippocampal neurons. Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.

Protein Kinase G Inhibits T‐type Ca2+ Channels in Rat Cerebral Arteries

In rat cerebral arteries, T‐type Ca2+ channels are suppressed by protein kinase A (PKA) signaling. In this study, we examine whether protein kinase G (PKG), another key vasodilatory pathway, exerts a similar or distinctive influence on this conductance. Using patch clamp electrophysiology and cerebral arterial smooth muscle cells from rat, we monitored an inward Ba2+ current that was divisible into nifedipine‐sensitive and –insensitive components. The nifedipine‐insensitive conductance displayed properties reminiscent of T‐type Ca2+ channels; faster activation and steady state inactivation particularly at hyperpolarized potentials. Intriguingly, agents that target key PKG signaling proteins altered T‐type Ca2+ channel activity. In particular, activators of PKG signaling (db‐cGMP, Na nitroprusside, SNAP) suppressed T‐type currents and evoked a hyperpolarized shift in steady state inactivation. PKG inhibitors (KT5823, ODQ) masked this suppression while having no effect on basal T‐type activity. In contrast, PKG manipulations had no influence on the nifedipine‐sensitive L‐type conductance. Cumulatively, our findings support the emerging view that T‐type Ca2+ channels are a key regulatory target of vasodilatory signaling pathways (i.e. PKG and PKA).

Ethanol Inhibition of a T‐Type Ca2+ Channel Through Activity of Protein Kinase C

T-type calcium channels (T-channels) are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol (EtOH) effects and the underlying mechanism of action in human embryonic kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans rats. Whole-cell voltage-clamp recordings were performed at 32 to 34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10-minute application of EtOH or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate [PMA]). We recorded T-type Ca²⁺ currents (T-currents) from 3 channel isoforms (CaV3.1, CaV3.2, and CaV3.3) before and during administration of EtOH. We found that only 1 isoform, CaV3.2, was significantly affected by EtOH. EtOH reduced current density as well as producing a hyperpolarizing shift in steady-state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T-currents from DRG neurons that are known to be enriched in CaV3.2. A myristoylated PKC peptide inhibitor (MPI) blocked the major EtOH effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of EtOH, while higher concentration PMA (1 μM) did not, suggesting that the EtOH effects operate through activation of PKC and were mimicked by lower concentration of PMA. EtOH primarily affects the CaV3.2 isoform of T-type Ca²⁺ channels acting through PKC, highlighting a novel target and mechanism for EtOH effects on excitable membranes.

Protein kinase a regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle

Recent investigations have identified that T-type Ca(2+) channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the CaV3.1/CaV3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba(2+) current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca(2+) channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni(2+), a concentration that selectively interferes with CaV3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the CaV3.2 subtype. In summary, this study revealed that a T-type Ca(2+) channel conductance can be isolated in arterial smooth muscle, and differentiated into CaV3.1 and CaV3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting would impact Ca(2+) dynamics and consequent tone regulation in the cerebral circulation.

Calcium Dysregulation of Voltage-Gated Sodium Channels Harboring LQT3 Mutations

Intracellular calcium ions modulate sodium channel inactivation by producing a depolarizing shift the steady-state inactivation equilibrium. We have recently proposed a mechanism for this effect by which direct Ca2+/calmodulin(CaM) binding to the inactivation gate increases the transient availability of channels in the action potential by shifting the steady-state inactivation. Interestingly, a crystal structure of Ca2+/CaM bound to the inactivation gate of the sodium channel pinpoints the position of four mutations (M1498T, K1500Δ, L1501V, G1502S) shown previously to underlie long QT3 syndrome, along a critical binding interface. We explored the possibility that these mutations, in addition to effects on channel gating, may alter calcium regulation of Nav1.5. This possibility was first tested directly with Isothermal Titration Calorimetry (ITC) to determine the binding parameters of purified proteins and then by patch-clamp electrophysiology of expressed wild-type and mutant channels. Interestingly, ITC experiments demonstrated that the mutations impacted Ca2+/CaM binding by altering the affinity of Ca2+/CaM for the inactivation gate. Channels carrying inherited mutation showed robust expression in HEK-293 cells with either modest or severe effects on channel gating, as expected for LQT3 mutations. However, in terms of Ca2+ regulation, the LQT3 mutations significantly altered the calcium-induced shift in steady-state inactivation compared to wild-type channels. The data suggest that calcium dysregulation of the voltage-gated sodium channel may contribute to the pathogenesis of LQT3 syndrome.