Erwin F. Shibata

Voltage-gated Nav channel targeting in the heart requires an ankyrin-G–dependent cellular pathway

Voltage-gated Nav channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Nav1.5 is the predominant Nav channel, and Nav1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Nav1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Nav channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Nav1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Nav1.5 expression, abnormal Nav1.5 membrane targeting, and reduced Na+ channel current density. We define the structural requirements on ankyrin-G for Nav1.5 interactions and demonstrate that loss of Nav1.5 targeting is caused by the loss of direct Nav1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Nav channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Nav channel organization in multiple excitable tissues.

Enhancement of rabbit cardiac sodium channels by beta-adrenergic stimulation.

Voltage-dependent sodium channels from a variety of tissues are known to be phosphorylated by the cAMP-dependent protein kinase, protein kinase A. However, the functional significance of sodium channel phosphorylation is not clearly understood. Using whole-cell voltage-clamp techniques, we show that sodium currents (INas) in rabbit cardiac myocytes are enhanced by isoproterenol (ISO). This enhancement of INa by ISO 1) is holding potential dependent, 2) can be mimicked by forskolin and dibutyryl cAMP, and 3) is accompanied by an increase in the rate of Na+ channel inactivation. In single-channel, inside-out patch experiments, the catalytic subunit of protein kinase A also enhances INa and increases the rate of inactivation, suggesting that cardiac Na+ channel phosphorylation may be physiologically important. Addition of the protein kinase A inhibitor to the pipette solution in whole-cell experiments blocks the stimulatory effect of forskolin without blocking the effect of ISO, suggesting that ISO also enhances INa through a cAMP-independent pathway. To determine if ISO may stimulate INa through a direct G protein pathway, single channels were recorded in the presence of the Gs-activating GTP analogue, GTP gamma S, and the stimulatory G protein subunit, Gs alpha. Both of these agents enhanced INa without affecting the rate of Na+ channel inactivation. These results suggest that ISO enhances rabbit cardiac INa through a dual (direct and indirect) G protein regulatory pathway.

Modulation of rat cardiac sodium channel by the stimulatory G protein α subunit

1 Modulation of cardiac sodium currents (INa) by the G protein stimulatory α subunit (Gsα) was studied using patch‐clamp techniques on freshly dissociated rat ventricular myocytes. 2 Whole‐cell recordings showed that stimulation of β‐adrenergic receptors with 10 μM isoprenaline (isoproterenol, ISO) enhanced INa by 68·4 ± 9·6 % (mean ±s.e.m.; n= 7, P < 0·05vs. baseline). With the addition of 22 μg ml−1 protein kinase A inhibitor (PKI) to the pipette solution, 10 μM ISO enhanced INa by 30·5 ± 7·0 % (n= 7, P < 0·05vs. baseline). With the pipette solution containing both PKI and 20 μg ml−1 anti‐Gsα IgG or 20 μg ml−1 anti‐Gsα IgG alone, 10 μM ISO produced no change in INa. 3 The effect of Gsα on INa was not due to changes in the steady‐state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation. 4 Whole‐cell INa was increased by 45·2 ± 5·3 % (n= 13, P < 0·05vs. control) with pipette solution containing 1 μM Gsα27‐42 peptide (amino acids 27‐42 of rat brain Gsα) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsα27‐42 to Na+ channels in inside‐out macropatches increased the ensemble‐averaged INa by 32·5 ± 6·8 % (n= 8, P < 0·05vs. baseline). The increase in INa was reversible upon Gsα27‐42 peptide washout. Single channel experiments showed that the Gsα27‐42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch. 5 Application of selected short amino acid segments (Gsα27‐36, Gsα33‐42 and Gsα30‐39) of the 16 amino acid Gsα peptide (Gsα27‐42 peptide) showed that only the C‐terminal segment of this peptide (Gsα33‐42) significantly increased INa in a dose‐dependent fashion. These results show that cardiac INa is regulated by Gsα via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33‐42) near the N‐terminus of Gsα is important in modulating cardiac Na+ channels.

A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells.

1. Voltage‐ and time‐dependent outward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit left descending coronary artery using a single pipette voltage clamp technique. The calcium‐activated potassium current was blocked by inclusion of EGTA in the pipette solution and CdCl2 in the extracellular bath. 2. Outward currents were elicited with depolarizing voltage steps to potentials positive to ‐20 mV. Long (5 s) voltage steps revealed slow inactivation of the current with a time constant of nearly 3 s at +60 mV. Potassium was identified as the predominant charge carrier by reversal potential measurements in potassium substitution experiments. 3. The results of kinetic analyses compared favourably with the Hodgkin‐Huxley model for a delayed rectifier with some deviations. The sigmoid current onset was best fitted by raising the activation variable (n) to the second power. Deactivation tail currents were consistently found to be comprised of two exponential components. The kinetics of activation and deactivation were strongly voltage‐dependent from ‐80 to +60 mV. 4. Envelope of tails experiments showed that the scaled tail current amplitudes followed the kinetic behaviour of current activation. The contribution of each of the two exponential tail components was also measured in these experiments. They did not reveal kinetically separable currents, nor were they differentially altered by 4‐aminopyridine (4‐AP), tetraethylammonium (TEA), or elevated [K+]o. 5. The steady‐state voltage‐dependence curves for both activation and inactivation were well fitted by a Boltzmann distribution with V1/2 = ‐5.60 mV and k = ‐8.66 mV for n infinity act and V1/2 = ‐24.20 mV and k = 5.16 mV for n infinity act. Super‐imposition of the two curves revealed a ‘window’ of voltage where channels are available for activation without completely inactivating. 6. Neither of the commonly used potassium channel blockers, TEA or 4‐AP, were particularly effective blockers of IK, reducing current by only 50‐70% at an extracellular concentration of 10 mM. TEA block was mildly voltage‐dependent and was more effective in reducing current towards the end of a 500 ms depolarization. 4‐AP, on the other hand, demonstrated considerable voltage‐dependence and preferentially reduced early currents. 7. Outward currents recorded from guinea‐pig and human coronary artery myocytes under the same conditions as in the rabbit cell experiments displayed similar characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes.

1 Whole‐cell Na+ currents (holding potential, −80 mV; test potential, −30 mV) in rat myocytes were inhibited by 8,9‐epoxyeicosatrienoic acid (8,9‐EET) in a dose‐dependent manner with 22 ± 4 % inhibition at 0.5 μM, 48 ± 5 % at 1 μM, and 73 ± 5 % at 5 μM (mean ± s.e.m., n= 10, P < 0.05 for each dose vs. control). Similar results were obtained with 5,6‐, 11,12‐, and 14,15‐EETs, while 8,9‐dihydroxyeicosatrienoic acid (DHET) was 3‐fold less potent and arachidonic acid was 10‐ to 20‐fold less potent. 2 8,9‐EET produced a dose‐dependent, hyperpolarized shift in the steady‐state membrane potential at half‐maximum inactivation (V½), without changing the slope factor. 8,9‐EET had no effect on the steady‐state activation of Na+ currents. 3 Inhibition of Na+ currents by 8,9‐EET was use dependent, and channel recovery was slowed. The effects of 8,9‐EET were greater at depolarized potentials. 4 Single channel recordings showed 8,9‐EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5 Incubation of cultured cardiac myocytes with 1 μM [3H]8,9‐EET showed that 25 % of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86 % of the radioactivity remained as [3H]8,9‐EET while 13 % was converted into [3H]8,9‐DHET. After a 30 min incubation, 1–2 % of the [3H]8,9‐EET uptake by cells remained as unesterified EET. 6 These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9‐EET. 8,9‐EET is a potent use‐ and voltage‐dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

Calcium-activated chloride current in rabbit coronary artery myocytes.

Whole-cell patch-clamp techniques were used to study enzymatically dispersed epicardial coronary artery smooth muscle cells. Depolarizing voltage pulses of 500-millisecond duration from -60 mV (118 mmol/L CsCl, 22 mmol/L tetraethylammonium chloride, and 5 mmol/L EGTA pipette solution) elicited inward L-type calcium currents (ICa). When EGTA was omitted from the pipette solution, an outward current was superimposed on the calcium current, and repolarizing voltage steps produced an inward tail current (IT). The amplitude of these inward currents was proportional to the ICa amplitude from -30 to +50 mV. The time course of decay of the current was well fit by a single exponential equation. The time constant (tau) of this equation did not change with the size of IT but was clearly voltage dependent (shorter at more negative potentials). Changing the chloride reversal potential from -1.3 to -39.7 mV by anion substitution using methanesulfonate as the chloride replacement in the pipette solution shifted the zero current level of IT from 0.9 +/- 0.56 to -33.1 +/- 0.85 mV. The tail current was blocked by nifedipine (10(-6) mol/L) and by isosmolar calcium substitution with barium in the bath solution and was enhanced by the dihydropyridine agonist Bay K 8644 (10(-6) mol/L). IT was also blocked by the chloride channel blockers DIDS (10(-4) mol/L) and niflumic acid (10(-5) mol/L). Caffeine (10(-2) mol/L), which releases intracellular calcium stores, caused an inward current at holding potentials (-60 mV), which was inhibited by DIDS. Caffeine also inhibited subsequent attempts to elicit IT by depolarizing pulses (88% reduction in IT).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium currents in isolated rabbit coronary arterial smooth muscle myocytes.

1. Calcium inward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit epicardial left descending coronary artery using a single‐pipette voltage‐clamp technique. Outward K+ currents were blocked with CsCl‐tetraethylammonium‐filled pipette solutions. 2. Relaxed coronary smooth muscle cells had a maximum diameter of 8.6 +/‐ 0.6 microns and a cell length of 96.7 +/‐ 3.3 microns when bathed in 2.5 mM [Ca2+]o. The average resting membrane potential at room temperature was ‐32 +/‐ 10 mV. The mean cell capacitance was 18.5 +/‐ 1.7 pF and the input resistance was 3.79 +/‐ 0.58 G omega. 3. Depolarizing voltage‐clamp steps from a holding potential of ‐80 mV elicited a single time‐ and voltage‐dependent inward current which was dependent upon extracellular [Ca2+]. In 2.5 mM [Ca2+]o, the inward current was activated at a potential of ‐40 mV and peaked at +10 mV. This current was inhibited by 0.5 mM‐CdCl2 and 1 microM‐nifedipine and was enhanced with 1 microM‐Bay K 8644. No detectable low‐threshold, rapidly inactivating T‐type calcium current was observed. 4. The apparent reversal potential of this inward current in 2.5 mM [Ca2+]o was +70 mV and shifted by 33.0 mV per tenfold increase in [Ca2+]o. This channel was also more permeable to barium and strontium ions than to calcium ions. 5. Single calcium channel recordings with 110 mM‐Ba2+ as the charge carrier revealed a mean slope conductance of 20.7 +/‐ 0.8 pS. 6. This calcium current (ICa) exhibited a strong voltage‐dependent inactivation process. However, the steady‐state inactivation curve (f infinity) displayed a slight nonmonotonic, U‐shaped dependence upon membrane potential. The potential at which half of the channels were inactivated was ‐27.9 mV with a slope factor of 6.9 mV. The steady‐state activation curve (d infinity) was also well‐described by a Boltzmann distribution with a half‐activation potential at ‐4.4 mV and a slope factor of ‐63 mV. ICa was fully activated at approximately +20 mV. 7. The rate of inactivation was dependent upon the species of ion carrying the current. Both Sr2+ and Ba2+ decreased the rate as well as the degree of inactivation. The tau f (fitted time constant of inactivation) curve displayed a U‐shaped relationship in 2.5 mM [Ca2+]o. The reactivation process was voltage dependent and could be described by a single exponential. 8. The current amplitude and the inactivation kinetics were temperature dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Autonomic Regulation of Voltage-Gated Cardiac Ion Channels

Altering voltage‐gated ion channel currents, by changing channel number or voltage‐dependent kinetics, regulates the propagation of action potentials along the plasma membrane of individual cells and from one cell to its neighbors. Functional increases in the number of cardiac sodium channels (NaV1.5) at the myocardial sarcolemma are accomplished by the regulation of caveolae by β adrenergically stimulated G‐proteins. We demonstrate that NaV1.5, CaV1.2a, and KV1.5 channels specifically localize to isolated caveolar membranes, and to punctate regions of the sarcolemma labeled with caveolin‐3. In addition, we show that NaV1.5, CaV1.2a, and KV1.5 channel antibodies label the same subpopulation of isolated caveolae. Plasma membrane sheet assays demonstrate that NaV1.5, CaV1.2a, and KV1.5 cluster with caveolin‐3. This may have interesting implications for the way in which adrenergic pathways alter the cardiac action potential morphology and the velocity of the excitatory wave.

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

Cardiac sodium channels (voltage-gated Na(+) channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying beta-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-alpha (G(s)alpha) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional G(s)alpha-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of G(s)alpha. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with beta-receptor agonists increased the sodium current (I(Na)) by 35.9 +/- 8.6% (P < 0.05), and this increase was mimicked by application of G(s)alpha protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that G(s)alpha and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of I(Na). In this study, alanine scanning mutagenesis of G(s)alpha (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of G(s)alpha (H41) is a critical residue for the functional increase of I(Na). Protein interaction assays suggest that G(s)alphaFL (full length) binds to caveolin-3, but the enhancement of I(Na) is observed only in the presence of G(s)alpha H41. We conclude that G(s)alpha H41 is a critical residue in the regulation of the increase in I(Na) in ventricular myocytes.

Threshold Effects of Acetylcholine on Primary Pacemaker Cells of the Rabbit Sino-Atrial Node

Leading or primary pacemaker cells located within the rabbit sino-atrial node have been identified by using electrophysiological and pharmacological techniques. Stable intracellular recordings lasting 20-30 min from cells within the s. a. node reveal three distinct patterns of spontaneous intracellular responses: (i) leading or primary pacing; (ii) follower or subsidiary pacing; and (iii) ‘anomalous’ pacemaker discharge. Our main objective was to measure the first detectable effect, or effects, of acetylcholine on the spontaneous intracellular electrical activity in mammalian primary pacemaker cells. Trains of brief ‘field’ stimuli were applied to evoke transmitter release from endogenous nerve varicosities. Systematic variations in the amplitude and duration of each stimulus, and in the train length; in conjunction with application of β blockers (l-pindolol (10-6 M); l-propranolol, (2 x 10-7 M)) yielded small and transient, but very consistent negative chronotropic effects. These electrophysiological changes were blocked by atropine (1 x 10-7 M) and were mimicked by bath application of low doses of acetylcholine (10-7-10-6 M) or muscarine chloride (10-8-10-7 M). In primary cells the first, or threshold effect of vagal excitation is a decrease in the slope of the pacem aker potential, without a detectable (less th an 2 mV) hyperpolarization or change in action potential duration. A reduction in the d V / dtmax of the initial depolarization is also quite consistently observed. Application of longer stimulus trains yield the classical hyperpolarizing response, which is often assumed to be the major electrophysiological correlate of the negative chronotropic effect. These data provide a detailed electrophysiological description of the ‘physiological’ effects of the vagus nerve excitation on primary or leading pacemaker cells of the mammalian s.-a. node. A plausible explanation for the absence of hyperpolarization is suggested; and a working hypothesis is presented for the changes in ionic current or currents, that underlie this negative chronotropic effect.