Abstract-This study demonstrates that caveolae, omega-shaped membrane invaginations, are involved in cardiac sodium channel regulation by a mechanism involving the ␣ subunit of the stimulatory heterotrimeric G-protein, G␣ s , via stimulation of the cell surface ␤-adrenergic receptor. Stimulation of ␤-adrenergic receptors with 10 mol/L isoproterenol in the presence of a protein kinase A inhibitor increased the whole-cell sodium current by a "direct" cAMP-independent G-protein mechanism. The addition of antibodies against caveolin-3 to the cell's cytoplasm via the pipette solution abrogated this direct G protein-induced increase in sodium current, whereas antibodies to caveolin-1 or caveolin-2 did not. Voltage-gated sodium channel proteins were found to associate with caveolin-rich membranes obtained by detergent-free buoyant density separation. The purity of the caveolar membrane fraction was verified by Western blot analyses, which indicated that endoplasmic/sarcoplasmic reticulum, endosomal compartments, Golgi apparatus, clathrin-coated vesicles, and sarcolemmal membranes were excluded from the caveolin-rich membrane fraction. Additionally, the sodium channel was found to colocalize with caveolar membranes by immunoprecipitation, indirect immunofluorescence, and immunogold transmission electron microscopy. These results suggest that stimulation of ␤-adrenergic receptors, and thereby G␣ s , promotes the presentation of cardiac sodium channels associated with caveolar membranes to the sarcolemma. Key Words: caveolae Ⅲ ion channel Ⅲ signal transduction Ⅲ cardiac Ⅲ adrenergic R egulation of voltage-gated ion channels at the plasma membrane of excitable cells is essential in maintaining cellular excitability and electrical impulse propagation. The amplitude and slope of the action potential upstroke are especially important in the control of cardiac conduction velocity, and the maintenance of appropriate waves of excitation through the ventricles. In the nonpacemaker cells of the heart, the voltage-gated sodium channel mediates the upstroke of the action potential. Neurohumoral regulation of this sodium current, I Na , via ␤-adrenergic stimulation has been shown to increase the current by at least 2 known mechanisms: one "direct" and one "indirect". [1][2][3][4][5][6][7] The indirect, or protein kinase A (PKA)-dependent, mechanism regulates I Na by phosphorylation of the channel protein at previously identified sites, 6 resulting in alterations of single-channel voltage-dependent characteristics, including channel availability (inactivation). 3,4,8 -12 The mechanism of the direct, PKA-independent, effect is not well understood, though evidence suggests that ligand binding to plasma membrane ␤-adrenergic receptors (␤ARs) results in the activation of a signaling cascade involving the G␣ s protein itself. This produces an increase in I Na without changes in single-channel characteristics or shifts in voltage-dependent current activation as demonstrated by current-voltage relationships. 2,7 In the presence of inhibito...

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.

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.

1. Modulation of cardiac sodium currents (INa) by the G protein stimulatory alpha subunit (Gsalpha) was studied using patch-clamp techniques on freshly dissociated rat ventricular myocytes. 2. Whole-cell recordings showed that stimulation of beta-adrenergic receptors with 10 microM isoprenaline (isoproterenol, ISO) enhanced INa by 68.4 +/- 9.6 % (mean +/- s.e.m.; n = 7, P < 0.05 vs. baseline). With the addition of 22 microgram ml-1 protein kinase A inhibitor (PKI) to the pipette solution, 10 microM ISO enhanced INa by 30.5 +/- 7.0 % (n = 7, P < 0.05 vs. baseline). With the pipette solution containing both PKI and 20 microgram ml-1 anti-Gsalpha IgG or 20 microgram ml-1 anti-Gsalpha IgG alone, 10 microM ISO produced no change in INa. 3. The effect of Gsalpha 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 < 0.05 vs. control) with pipette solution containing 1 microM Gsalpha27-42 peptide (amino acids 27-42 of rat brain Gsalpha) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsalpha27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged INa by 32.5 +/- 6.8 % (n = 8, P < 0.05 vs. baseline). The increase in INa was reversible upon Gsalpha27-42 peptide washout. Single channel experiments showed that the Gsalpha27-42 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 (Gsalpha27-36, Gsalpha33-42 and Gsalpha30-39) of the 16 amino acid Gsalpha peptide (Gsalpha27-42 peptide) showed that only the C-terminal segment of this peptide (Gsalpha33-42) significantly increased INa in a dose-dependent fashion. These results show that cardiac INa is regulated by Gsalpha 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 Gsalpha is important in modulating cardiac Na+ channels.

Arachidonic acid is a precursor of many bioactive lipids that are involved in signal transduction and cellular regulatory mechanisms. In addition to the well-known and wellestablished cyclo-oxygenase and lipoxygenase pathways which generate important bio-mediators such as prostaglandins, thromboxanes and leukotrienes, the cytochrome P450 monoxygenase pathway has also emerged as an important source of bioactive arachidonic acid derivatives (McGiff, 1991). Cytochrome P450 monooxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers : 5,6-, 8,9-, 11,12-and 14,15_EET, as well as to 19-and 20-hydroxyeicosatetraenoic acids (Oliw, 1994). EETs are potent endothelium-derived vasodilators that modulate vascular tone via enhancement of Ca¥-activated K¤ channels in vascular smooth muscle, suggesting that these compounds are endothelium-derived hyperpolarizing factors (Gebremedhin