An emerging antiarrhythmic target: late sodium current.

The cardiac late sodium current (INa,L) has been in the focus of research in the recent decade. The first reports on the sustained component of voltage activated sodium current date back to the seventies, but early studies interpreted this tiny current as a product of a few channels that fail to inactivate, having neither physiologic nor pathologic implications. Recently, the cardiac INa,L has emerged as a potentially major arrhythmogenic mechanism in various heart diseases, attracting the attention of clinicians and researchers. Research activity on INa,L has exponentially increased since Ranolazine, an FDA-approved antianginal drug was shown to successfully suppress cardiac arrhythmias by inhibiting INa,L. This review aims to summarize and discuss a series of papers focusing on the cardiac late sodium current and its regulation under physiological and pathological conditions. We will discuss critical evidences implicating INa,L as a potential target for treating myocardial dysfunction and cardiac arrhythmias.

Vasoactive intestinal peptide-stimulated Cl- secretion: activation of cAMP-dependent K+ channels.

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.

Large currents generate cardiac Ca2+ sparks.

Previous models of cardiac Ca2+ sparks have assumed that Ca2+ currents through the Ca2+ release units (CRUs) were approximately 1-2 pA, producing sparks with peak fluorescence ratio (F/F(0)) of approximately 2.0 and a full-width at half maximum (FWHM) of approximately 1 microm. Here, we present actual Ca2+ sparks with peak F/F(0) of >6 and a FWHM of approximately 2 microm, and a mathematical model of such sparks, the main feature of which is a much larger underlying Ca2+ current. Assuming infinite reaction rates and no endogenous buffers, we obtain a lower bound of approximately 11 pA needed to generate a Ca2+ spark with FWHM of 2 microm. Under realistic conditions, the CRU current must be approximately 20 pA to generate a 2- microm Ca2+)spark. For currents > or =5 pA, the computed spark amplitudes (F/F(0)) are large (approximately 6-12 depending on buffer model). We considered several factors that might produce sparks with FWHM approximately 2 microm without using large currents. Possible protein-dye interactions increased the FWHM slightly. Hypothetical Ca2+ "quarks" had little effect, as did blurring of sparks by the confocal microscope. A clusters of CRUs, each producing 10 pA simultaneously, can produce sparks with FWHM approximately 2 microm. We conclude that cardiac Ca2+ sparks are significantly larger in peak amplitude than previously thought, that such large Ca2+ sparks are consistent with the measured FWHM of approximately 2 microm, and that the underlying Ca2+ current is in the range of 10-20 pA.

Evolution of cardiac calcium waves from stochastic calcium sparks.

We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.

G(i)-dependent localization of beta(2)-adrenergic receptor signaling to L-type Ca(2+) channels.

A plausible determinant of the specificity of receptor signaling is the cellular compartment over which the signal is broadcast. In rat heart, stimulation of beta(1)-adrenergic receptor (beta(1)-AR), coupled to G(s)-protein, or beta(2)-AR, coupled to G(s)- and G(i)-proteins, both increase L-type Ca(2+) current, causing enhanced contractile strength. But only beta(1)-AR stimulation increases the phosphorylation of phospholamban, troponin-I, and C-protein, causing accelerated muscle relaxation and reduced myofilament sensitivity to Ca(2+). beta(2)-AR stimulation does not affect any of these intracellular proteins. We hypothesized that beta(2)-AR signaling might be localized to the cell membrane. Thus we examined the spatial range and characteristics of beta(1)-AR and beta(2)-AR signaling on their common effector, L-type Ca(2+) channels. Using the cell-attached patch-clamp technique, we show that stimulation of beta(1)-AR or beta(2)-AR in the patch membrane, by adding agonist into patch pipette, both activated the channels in the patch. But when the agonist was applied to the membrane outside the patch pipette, only beta(1)-AR stimulation activated the channels. Thus, beta(1)-AR signaling to the channels is diffusive through cytosol, whereas beta(2)-AR signaling is localized to the cell membrane. Furthermore, activation of G(i) is essential to the localization of beta(2)-AR signaling because in pertussis toxin-treated cells, beta(2)-AR signaling becomes diffusive. Our results suggest that the dual coupling of beta(2)-AR to both G(s)- and G(i)-proteins leads to a highly localized beta(2)-AR signaling pathway to modulate sarcolemmal L-type Ca(2+) channels in rat ventricular myocytes.

Theoretical analysis of the Ca2+ spark amplitude distribution.

A difficulty of using confocal microscopy to study Ca2+ sparks is the uncertainty of the linescan position with respect to the source of Ca2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca2+ spark amplitudes (a) even if all Ca2+ sparks were generated identically. Thus variations in Ca2+ spark amplitude due to positional differences between confocal linescans and Ca2+ release site are intertwined with variations due to intrinsic differences in Ca2+ release properties. To separate these two sources of variations on the Ca2+ spark amplitude, we determined the effect changes of channel current or channel open time--collectively called the source strength, alpha--had on the measured Ca2+ spark amplitude histogram, N(a). This was done by 1) simulating Ca2+ release, Ca2+ and fluo-3 diffusion, and Ca2+ binding reactions; 2) simulation of image formation of the Ca2+ spark by a confocal microscope; and 3) using a novel automatic Ca2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f alpha (alpha), to N(a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca(2+)-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f alpha (alpha) can, in principle, be calculated from the Ca2+ spark amplitude histogram since N(a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f alpha (alpha). In particular, if all Ca2+ sparks were generated identically, then the plot of 1/N(a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/N(a) plot. 3) Although the inverse relationship between Ca2+ spark amplitude and decay time might be used to distinguish Ca2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca2+ sparks.

A class of parametrically excited calcium oscillation detectors.

Intracellular Ca2+ oscillations are often a response to external signals such as hormones. Changes in the external signal can alter the frequency, amplitude, or form of the oscillations suggesting that information is encoded in the pattern of Ca2+ oscillations. How might a cell decode this signal? We show that an excitable system whose kinetic parameters are modulated by the Ca2+ concentration can function as a Ca2+ oscillation detector. Such systems have the following properties: (1) They are more sensitive to an oscillatory than to a steady Ca2+ signal. (2) Their response is largely independent of the signal amplitude. (3) They can extract information from a noisy signal. (4) Unlike other frequency sensitive detectors, they have a flat frequency response. These properties make a Ca(2+)-sensitive excitable system nearly ideal for detecting and decoding Ca2+ oscillations. We suggest that Ca2+ oscillations, in concert with these detectors, can act as cellular timekeepers to coordinate related biochemical reactions and enhance their overall efficiency.

Chronic ethanol exposure inhibits ATP-stimulated but not KCl-stimulated dopamine release in PC 12 cells.

The functional significance of the ethanol-induced alterations in intracellular concentration of free calcium ([Ca++]i) was determined in PC 12 cells by measuring agonist-stimulated dopamine (DA) release after ethanol exposure. ATP and KCl produced a concentration-dependent release of DA, which was linearly related to the net increase in [Ca++]i, but different relationships were observed with ATP and KCl. Acute addition of 150 mM ethanol inhibited KCl-stimulated release of DA, but did not alter the response to ATP. In contrast, a 4-day exposure to 150 mM ethanol led to a reduction in ATP-evoked DA release without altering the response to KCl. Furthermore, a 7-day treatment with 25 mM ethanol also decreased the response to ATP. Acute and chronic ethanol exposures, however, did not alter the relationships between DA release and the increase in [Ca++]i observed with ATP and KCl. The data indicate that acute and chronic ethanol treatments have differential effects on the responses to extracellular ATP and KCl and alter DA release primarily by altering the calcium influx stimulated by agonists.