Negative Shift In Voltage Dependence Research Articles

Anti-arrhythmic potential of the late sodium current inhibitor GS-458967 in murine Scn5a-1798insD+/- and human SCN5A-1795insD+/- iPSC-derived cardiomyocytes.

Selective inhibition of cardiac late sodium current (INaL) is an emerging target in the treatment of ventricular arrhythmias. We investigated the electrophysiological effects of GS-458967 (GS967), a potent, selective inhibitor of INaL, in an overlap syndrome model of both gain and loss of sodium channel function, comprising cardiomyocytes derived from both human SCN5A-1795insD+/- induced pluripotent stem cells (hiPSC-CMs) and mice carrying the homologous mutation Scn5a-1798insD+/-. On patch-clamp analysis, GS967 (300 nmol/l) reduced INaL and action potential (AP) duration in isolated ventricular myocytes from wild type and Scn5a-1798insD+/- mice, as well as in SCN5A-1795insD+/- hiPSC-CMs. GS967 did not affect the amplitude of peak INa, but slowed its recovery, and caused a negative shift in voltage-dependence of INa inactivation. GS967 reduced AP upstroke velocity in Scn5a-1798insD+/- myocytes and SCN5A-1795insD+/- hiPSC-CMs. However, the same concentration of GS967 did not affect conduction velocity in Scn5a-1798insD+/- mouse isolated hearts, as assessed by epicardial mapping. GS967 decreased the amplitude of delayed after depolarizations and prevented triggered activity in mouse Scn5a-1798insD+/- cardiomyocytes. The INaL inhibitor GS967 decreases repolarization abnormalities and has anti-arrhythmic effects in the absence of deleterious effects on cardiac conduction. Thus, selective inhibition of INaL constitutes a promising pharmacological treatment of cardiac channelopathies associated with enhanced INaL. Our findings furthermore implement hiPSC-CMs as a valuable tool for assessment of novel pharmacological approaches in inherited sodium channelopathies.

Fly DPP10 acts as a channel ancillary subunit and possesses peptidase activity

Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K+ channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides.

Inhibition of voltage-gated sodium channels by sumatriptan bioisosteres.

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7–1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)-22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that use-dependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors.

A Novel Cardiac Nav1.5 Channel Mutation, L812Q, Leads to Brugada Syndrome

Brugada syndrome (BS) is a genetically determined cardiac electrical disorder, characterized by typical electrocardiography (ECG) alterations, and it is an arrhythmogenic syndrome that may lead to sudden cardiac death. The most common genotype found among Brugada syndrome patients is caused by mutations in the SCN5A gene, which lead to a loss of function of the cardiac sodium (Na+) channel (Nav1.5) by different mechanisms. Here, we report that a novel nonsense SCN5A mutation, L812Q, localized in the transmembrane region DII-S4 of the Nav1.5 channel, was identified in an index patient who showed a Brugada syndrome type 1 ECG phenotype. The mutation was absent in the patient's parents and brother. Expression of the wild-type (WT) and L812Q mutant Nav1.5 channels in human embryonic kidney cells (HEK293 cells) reveals that the mutation result in a reduction of Na+ current density and a ∼20 mV negative shift of the voltage dependence of inactivation. However, the voltage dependence of activation and the time course for recovery from inactivation are not altered by the mutation. The negative shift of the steady-state inactivation curve reduced the Na+ window current. In addition, western blot and confocal laser microscopyimaging experiments suggest that the mutant channels may be retained in the endoplasmic reticulum, resulting in less channel expression at the membrane surface. Decreased membrane surface expression, a negative shift of voltage dependence of inactivation, and a decline of Na+ window current in the L812Q mutant may lead to a reduction of Na+ current during the upstroke of a cardiac action potential and an inhibition of the reopening of inactivated Na+ channels during the repolarization phases, all of which may contribute to the development of Brugada syndrome.

Oral Proton Pump Inhibitors Disrupt Horizontal Cell-Cone Feedback and Enhance Visual Hallucinations in Macular Degeneration Patients

Visual hallucinations (VHs) occur in macular degeneration patients with poor vision but normal cognitive function. The underlying mechanisms are poorly understood. We report the identification of pharmaceutical agents that enhance VH and use these agents to examine the contribution of retinal neurons to this syndrome. We detail clinical observations on VH in five macular degeneration patients treated with proton pump inhibitors having the core structure, 2-pyridyl-methylsulfinyl-benzimidazole. We tested possible retinal mechanisms using paired whole cell recordings to examine effects of these compounds on feedback interactions between horizontal cells and cones in amphibian retina. Five patients with advanced wet macular degeneration described patterned VHs that were induced or enhanced by oral proton pump inhibitors. The abnormal images increased with light, disappeared in the dark, and originated in the retina, based on ophthalmodynamometry. Simultaneous paired whole cell recordings from amphibian cones and horizontal cells showed that 2-pyridyl-methylsulfinyl-benzimidazoles blocked the negative shift in voltage dependence and increase in amplitude of the calcium current (ICa) in cones that is induced by changes in horizontal cell membrane potential. These effects disrupt the negative feedback from horizontal cells to cones that is important for the formation of center-surround receptive fields in bipolar and ganglion cells, and thus for normal spatial and chromatic perception. Our study suggests that changes in the output of retinal neurons caused by disturbances in outer retinal feedback mechanisms can enhance patterned visual hallucinations.

Cerebellar Ataxia by Enhanced CaV2.1 Currents Is Alleviated by Ca2+-Dependent K+-Channel Activators inCacna1aS218LMutant Mice

Mutations in the CACNA1A gene are associated with neurological disorders, such as ataxia, hemiplegic migraine, and epilepsy. These mutations affect the pore-forming α(1A)-subunit of Ca(V)2.1 channels and thereby either decrease or increase neuronal Ca(2+) influx. A decreased Ca(V)2.1-mediated Ca(2+) influx has been shown to reduce the regularity of cerebellar Purkinje cell activity and to induce episodic cerebellar ataxia. However, little is known about how ataxia can be caused by CACNA1A mutations that increase the Ca(2+) influx, such as the S218L missense mutation. Here, we demonstrate that the S218L mutation causes a negative shift of voltage dependence of Ca(V)2.1 channels of mouse Purkinje cells and results in lowered thresholds for somatic action potentials and dendritic Ca(2+) spikes and in disrupted firing patterns. The hyperexcitability of Cacna1a(S218L) Purkinje cells was counteracted by application of the activators of Ca(2+)-dependent K(+) channels, 1-EBIO and chlorzoxazone (CHZ). Moreover, 1-EBIO also alleviated the irregularity of Purkinje cell firing both in vitro and in vivo, while CHZ improved the irregularity of Purkinje cell firing in vitro as well as the motor performance of Cacna1a(S218L) mutant mice. The current data suggest that abnormalities in Purkinje cell firing contributes to cerebellar ataxia induced by the S218L mutation and they advocate a general therapeutic approach in that targeting Ca(2+)-dependent K(+) channels may be beneficial for treating ataxia not only in patients suffering from a decreased Ca(2+) influx, but also in those suffering from an increased Ca(2+) influx in their Purkinje cells.

M channel enhancers and physiological M channel block.

M-type (Kv7, KCNQ) K(+) channels control the resting membrane potential of many neurons, including peripheral nociceptive sensory neurons. Several M channel enhancers were suggested as prospective analgesics, and targeting M channels specifically in peripheral nociceptors is a plausible strategy for peripheral analgesia. However, receptor-induced inhibition of M channels in nociceptors is often observed in inflammation and may contribute to inflammatory pain. Such inhibition is predominantly mediated by phospholipase C. We investigated four M channel enhancers (retigabine, flupirtine, zinc pyrithione and H(2)O(2)) for their ability to overcome M channel inhibition via two phospholipase C-mediated mechanisms, namely depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a rise in intracellular Ca(2+) (an action mediated by calmodulin). Data from overexpressed Kv7.2/Kv7.3 heteromers and native M currents in dorsal root ganglion neurons suggest the following conclusions. (i) All enhancers had a dual effect on M channel activity, a negative shift in voltage dependence and an increase of the maximal current at saturating voltages. The enhancers differed in their efficacy to produce these effects. (ii) Both PIP(2) depletion and Ca(2+)/calmodulin strongly reduced the M current amplitude; however, at voltages near the threshold for M channel activation (-60 mV) all enhancers were able to restore M channel activity to a control level or above, while at saturating voltages the effects were more variable. (iii) Receptor-mediated inhibition of M current in nociceptive dorsal root ganglion neurons did not reduce the efficacy of retigabine or flupirtine to hyperpolarize the resting membrane potential. In conclusion, we show that all four M channel enhancers tested could overcome both PIP(2) and Ca(2+)-calmodulin-induced inhibition of Kv7.2/7.3 at voltages close to the threshold for action potential firing (-60 mV) but generally had reduced efficacy at a saturating voltage (0 mV). We suggest that the efficacy of an M channel enhancer to shift the voltage dependence of activation may be most important for rescuing M channel function in sensory neurons innervating inflamed tissue.

BK Channel Modulation by Leucine-Rich Repeat Containing Proteins

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Regulation by variable auxiliary subunits is a major mechanism to generate tissue- or cell-specific diversity of ion channel function.Mammalian large-conductance, voltage and calcium-activated potassium (BK, KCa1.1) channels are ubiquitously expressed with diverse functions in different tissues or cell types, consisting of the pore-forming, voltage- and Ca2+-sensing α-subunits (BKα), or together with the tissue-specific auxiliary subunits, previously known as four β-subunits (β1 - β4). We previously identified a leucine-rich repeat (LRR) containing protein, LRRC26, as a new type of BK channel auxiliary subunit, which causes an unprecedented large negative shift in voltage dependence of channel activation. Here we report a group of LRRC26 paralogous proteins LRRC52, LRRC55 and LRRC38 that potentially function as LRRC26-type auxiliary subunits of BK channels. LRRC52, LRRC55 and LRRC38 produce marked shifts in the BK channel's voltage dependence of activation in hyperpolarization direction to different extents. They together with LRRC26 show distinct expression in different human tissues and may have a broad influence of the BK channel function in different tissues or cell types.

High cortical spreading depression susceptibility and migraine‐associated symptoms in Cav2.1 S218L mice

The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

The Interaction of Antrhacene-9-Carboxylic Acid with Calcium-Activated Chloride Channels is Influenced by the State of Global Phosphorylation in Pulmonarty Artery Smooth Muscle Cells

Ca2+-dependent Cl- currents (IClCa) are inhibited by phosphorylation in arterial smooth muscle cells. We recently reported that niflumic acid (NFA), an inhibitor of IClCa, is less efficacious at blocking these currents in conditions promoting phosphorylation (Wiwchar et al., Br J Pharmacol, in press, 2009). This study aimed to assess whether another Cl- channel blocker, anthracene-9-carboxylic acid (A9C), is also affected by channel phosphorylation. A9C blocks IClCa at positive potentials but paradoxically stimulates the inward IClCa tail after repolarization to negative potentials (Piper & Greenwood, Br J Pharmacol 138: 31-38, 2003). IClCa was evoked by pipette solutions containing 500 nM free Ca2+ with or without 5 mM ATP to alter the state of phosphorylation. Although A9C (1-500 uM) dose-dependently blocked steady state IClCa at potentials positive to 0 mV in all cell groups, its maximal effect and sensitivity to voltage were enhanced in cells dialyzed with 0 vs. 5 mM ATP. For example, maximal block by 100 uM A9C was 35 and 73%, and V0.5 was 110 and 67 mV, in cells with 5 vs 0 ATP, respectively. A9C enhanced IClCa tail at −80 mV by causing a negative shift in voltage-dependence in both cell groups, with a larger shift occurring in cells dialyzed with 5 mM ATP. Interestingly, 100 but not 500 uM A9C stimulated steady-state ICl(Ca) at potentials < 0 mV in cells dialyzed with 0 ATP, a potential range where ICl(Ca) was unaffected in myocytes dialyzed with 5 mM ATP. As with NFA, the complex actions of A9C on IClCa are influenced by the state of channel phosphorylation and we propose the existence of at least two binding sites with different affinities for A9C.

Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function

Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

Dilated Cardiomyopathy due to Sodium Channel Dysfunction

The cardiac sodium channel mediates the rapid upstroke of the cardiac action potential and thereby constitutes a critical determinant of cardiac excitability and conduction. Mutations in the SCN5A gene encoding the α-subunit of this channel have been linked to a broad clinical spectrum of arrhythmia disorders, including long QT syndrome, Brugada syndrome, sick sinus syndrome, conduction disease, and most recently, atrial fibrillation.1,2 These primary arrhythmia syndromes were originally considered pure electrical entities occurring in the absence of structural heart disease, and the presence of myocardial abnormalities in these disorders was originally actually excluded by definition. Evidence is now accumulating, however, that sodium channelopathy can also be associated with the development of cardiac fibrosis, dilatation, and hypertrophy.3–8 Such structural changes within the myocardium may in turn further predispose to the development of ventricular arrhythmias. These findings have led to reevaluation of the initial view that mutations in a cardiac ion channel would only lead to pure electrical dysfunction and raised the intriguing possibility that the structural alterations could be a direct consequence of sodium channel dysfunction rather than a consequence of long-standing arrhythmia. The mechanism by which a dysfunctional sodium channel leads to structural changes in myocardial tissue, however, remains unclear. Article p 83 In this issue of Circulation: Arrhythmia and Electrophysiology , Ge et al8 report on a novel SCN5A mutation (A1180V) linked to dilated cardiomyopathy (DCM), expanding the repertoire of SCN5A mutations associated with DCM. Previously, the D1275N mutation was discovered independently by McNair et al5 and Olson et al6 in the same large family with the syndrome of DCM, atrial arrhythmias, sinus node dysfunction, and conduction disease. This family had been originally described by Greenlee et al9 in 1986. The D1275N mutation has also been associated with mild ventricular structural alterations in …

Blockade of HERG K+ channel by an antihistamine drug brompheniramine requires the channel binding within the S6 residue Y652 and F656

A number of clinically used drugs block delayed rectifier K+ channels and prolong the duration of cardiac action potentials associated with long QT syndrome. This study investigated the molecular mechanisms of voltage-dependent inhibition of human ether-a-go-go-related gene (HERG) delayed rectifier K+ channels expressed in HEK-293 cells by brompheniramine, an antihistamine. Brompheniramine inhibited HERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) value of 1.7 microm at 0 mV. A block of HERG current by brompheniramine was enhanced by progressive membrane depolarization and showed significantly negative shift in voltage-dependence of channel activation. Inhibition of HERG current by brompheniramine showed time-dependence. The S6 residue HERG mutant Y652A and F656C largely reduced the blocking potency of HERG current. These results indicate that brompheniramine mainly inhibited the HERG potassium channel through the residue Y652 and F656 and these residues may be an obligatory determinant in inhibition of HERG current for brompheniramine.

Insulin-Like Growth Factor-1 and PTEN Deletion Enhance Cardiac L-Type Ca 2+ Currents via Increased PI3Kα/PKB Signaling

Ca2+ influx through the L-type Ca2+ channel (I(Ca,L)) is a key determinant of cardiac contractility and is modulated by multiple signaling pathways. Because the regulation of I(Ca,L) by phosphoinositide-3-kinases (PI3Ks) and phosphoinositide-3-phosphatase (PTEN) is unknown, despite their involvement in the regulation of myocardial growth and contractility, I(Ca,L) was recorded in myocytes isolated from mice overexpressing a dominant-negative p110alpha mutant (DN-p110alpha) in the heart, lacking the PI3Kgamma gene (PI3Kgamma(-/-)) or with muscle-specific ablation of PTEN (PTEN(-/-)). Combinations of these genetically altered mice were also examined. Although there were no differences in the expression level of CaV1.2 proteins, basal I(Ca,L) densities were larger (P<0.01) in PTEN(-/-) myocytes compared with littermate controls, PI3Kgamma(-/-), or DN-p110alpha myocytes and showed negative shifts in voltage dependence of current activation. The I(Ca,L) differences seen in PTEN(-/-) mice were eliminated by pharmacological inhibition of either PI3Ks or protein kinase B (PKB) as well as in PTEN(-/-)/DN-p110alpha double mutant mice but not in PTEN(-/-)/PI3Kgamma(-/-) mice. On the other hand, application of insulin-like growth factor-1 (IGF-1), an activator of PKB, increased I(Ca,L) in control and PI3Kgamma(-/-), while having no effects on I(Ca,L) in DN-p110alpha or PTEN(-/-) mice. The I(Ca,L) increases induced by IGF-1 were abolished by PKB inhibition. Our results demonstrate that IGF-1 treatment or inactivation of PTEN enhances I(Ca,L) via PI3Kalpha-dependent increase in PKB activation.

Tyrosine kinase inhibition differentially regulates heterologously expressed HCN channels

The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na(+) and K(+). There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current i(f). Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phosphorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node i(f) conductance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages near the midpoint of activation. In the case of HCN2 there was also a negative shift in the voltage dependence of activation that accompanies the current reduction. We have shown previously that HCN2 is the dominant isoform expressed in rat ventricular myocytes. The above results predict that genistein should reduce i(f) in the rat ventricle and cause a negative shift of voltage dependence and kinetics of activation. We tested this hypothesis by studying the effects of genistein on isolated rat ventricular myocytes. Genistein significantly reduced i(f) current density (pA/pF) (control: 12.2+/-1.8; genistein: 3.5+/-0.5; washout: 7.7+/-0.8; n=10), and caused a negative shift of the midpoint of activation by 14 mV (-133+/-1 mV for genistein and -119+/-1 mV for washout, n=7) with no change in slope factor. Our results thus suggest that i(f) in the heart and i(f)-like currents in other tissues can be regulated differentially by tyrosine phosphorylation based on isoform expression patterns.

Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families.

One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na+ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (Ipst) at -20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8+/-0.2% and 1.9+/-0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The -10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by approximately 10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current.

Relation Between Bicarbonate Concentration and Voltage Dependence of Sodium Currents in Freshly Isolated CA1 Neurons of the Rat

It recently has been shown that whole cell calcium and sodium currents are modulated by CO(2)/HCO(3)(-)-buffered saline. While the bicarbonate ion, but not CO(2), has been proven to modulate calcium currents, this information is lacking for sodium currents. Furthermore, it is not known whether the strength of modulation dependents on the bicarbonate concentration or whether it is an all-or-nothing phenomenon. To answer these questions, we used the whole cell voltage-clamp technique on freshly isolated hippocampal CA1 neurons from the rat. A voltage step from -130 to -20 mV elicited a sodium current with an amplitude of -5.1 +/- 0.5 nA (mean +/- SE, n = 17) when cells were superfused with HEPES-buffered saline. The amplitude of this current increased during a subsequent superfusion with solutions containing increasing amounts of bicarbonate and CO(2) (%CO(2)/mM HCO(3)(-): 2.5/5.6; 5.0/18; 10/37), with a maximal increment in 10% CO(2)/37 mM HCO(3)(-) of -6.9 +/- 0.8 nA. The increase in amplitude was associated with a linear negative shift (slope: -0.7 mV/mM HCO(3)(-)) of the potential of half-maximal activation (DeltaV(h,a): -19.4 +/- 1.8 mV in 10% CO(2)) but not with an alteration in the maximal conductance (g(max): HEPES: 203.1 +/- 21.0 nS and 10% CO(2)/37 mM HCO(3)(-): 207.3 +/- 21.3 nS). In addition, the potential of half-maximal inactivation (V(h,i)) shifted to more negative potentials (slope: -0.6 mV/mM HCO(3)(-)) with increasing amounts of bicarbonate and CO(2) (HEPES: -53.6 +/- 11.8 mV; 10% CO(2)/37 mM HCO(3)(-): -69.8 +/- 2.1 mV), making the amplitude of the current highly sensitive for small potential changes at resting membrane potential. The same negative shift in voltage dependence arose when cells were exposed to solutions with different amounts of bicarbonate (5.6; 18; 26 mM) but constant CO(2) (5%) with slope rates of -0.5 mV/mM HCO(3)(-) for V(h,a) and -0.5 mV/mM HCO(3)(-) for V(h,i). Again, there was no correlation between bicarbonate concentration and the size of g(max). When currents were evoked in solutions containing a constant concentration (18 mM) of bicarbonate but different amounts of CO(2) (2.5; 5.0 10%), no significant changes have been observed. The present data demonstrate that bicarbonate ions, and not CO(2), modulate voltage-gated sodium currents in a concentration-dependent manner. Because the amplitude of the sodium current becomes highly sensitive to membrane potential changes concomitant with increased bicarbonate amounts, this may be critical for the excitability of the neuronal network in situations (like metabolic acidosis, respiratoric alkalosis and hypercapnia) in which the concentration of this ion can alter.

Voltage-Dependent Sodium Channel Function Is Regulated Through Membrane Mechanics

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na + channel α subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na + channels involve mechanical destabilization. Our observation that the β1 subunit causes similar changes in function of the Na + channel α subunit suggests that β1 may act through interaction with cytoskeleton.

Properties of sodium and potassium currents of cultured adult human atrial myocytes.

Cultured cell systems are valuable for the study of regulation of phenotypic expression, but little is known about the electrophysiological properties of human cardiac tissues in culture. The present studies were designed to determine the feasibility of maintaining human atrial myocytes in primary culture and to assess changes in Na+ (INa) and K+ (Ito, transient outward, and Ikur, ultra-rapid delayed rectifier) currents. Within 24 h of culture, cells assumed an avoid shape, which they maintained for up to 7 days. The voltage dependence, kinetics, and density of INa were unchanged in culture. The activation properties of Ito (kinetics and voltage dependence) were not altered, but Ito density (current normalized to cell capacitance) was reduced and inactivation properties were altered (negative shift in voltage dependence and slowed kinetics) in cultured compared with fresh cells. The absolute current amplitude, kinetics, voltage dependence, and 4-aminopyridine sensitivity of IKur were unchanged, but current density was increased. All changes in ionic currents occurred within 24 h of culture and remained stable for the next 4 days. We conclude that human atrial myocytes can be maintained in primary culture, that the qualitative properties of INa, Ito, and IKur remain constant but that some quantitative changes occur, and that cultured human atrial myocytes may be valuable for studies of the molecular mechanisms and regulation of cardiac channel function in humans.

Role of cyclic amp dependent protein kinase in cannabinoid receptor modulation of potassium “A-current” in cultured rat hippocampal neurons

Cannabinoid receptor agonists have been previously shown to enhance a potassium A-current (I A) in cultured rat hippocampal neurons. This effect has been further demonstrated to be dependent on G-protein linkage to adenylyl cyclase and levels of intracellular cyclic AMP (cAMP). The present study extends this analysis to the involvement of cAMP-dependent protein kinase (PKA) in this cascade. Specific activators and inhibitors of PKA were shown to have differential effects on the voltage dependence of I A. Specific activators of PKA produced a negative shift in voltage dependence of I A, whereas PKA inhibitors produced a positive shift in I A voltage dependence, the latter similar to that effected by the cannabinoid agonist WIN 55,212-2. Although the negative shift in I A induced by PKA stimulation could be reversed by PKA inhibitors, the positive shift produced by the PKA inhibitors alone was only 50–60% of the cannabinoid-produced shift in I A voltage dependence. This partial effect of PKA inhibition was confirmed by biochemical assays in the same cultured neurons that showed a similar 50–60% decrement in in vitro protein phosphorylation produced by PKA inhibitors. Results are discussed in terms of a diffusible second messenger linkage of the cannabinoid receptor to the A-current channel via the role of protein phosphorylation in modulation of I A.