High [Na+]i in cardiomyocytes from rainbow trout

Intracellular Na(+)-concentration, [Na(+)](i) modulates excitation-contraction coupling of cardiac myocytes via the Na(+)/Ca(2+) exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca(2+) influx via reverse-mode NCX contributes significantly to contraction when [Na(+)](i) is 16 mM but not 10 mM. However, physiological [Na(+)](i) has never been measured. We recorded [Na(+)](i) using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na(+)](i) at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21 degrees C). Surprisingly, we found that [Na(+)](i) was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na(+)](i) in resting cells. Thus, we report a resting in vivo [Na(+)](i) of 13.4 mM for rainbow trout cardiomyocytes. [Na(+)](i) increased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na(+)](i). Our results provide the first measurement of [Na(+)](i) in rainbow trout cardiomyocytes. This surprisingly high [Na(+)](i) is likely to result in physiologically significant Ca(2+) influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca(2+)-source will decrease with the action potential duration as temperature and contraction frequency increases.     

Enhancing effects of diethyldithiocarbamate on increase of sodium channel by sulfur dioxide derivatives in ventricular myocytes

The enhancing effects of diethyldithiocarbamate (DDC) on increase of sodium channel by sulfur dioxide derivatives in ventricular myocytes were studied using the whole cell patch-clamp technique to probe the mechanism of SO(2) on the cardiovascular system in this study. Firstly, the effects of DDC and/or sulfur dioxide (SO(2)) derivatives on the activities of superoxide dismutase (SOD) were studied. The results showed that DDC decreased SOD activities significantly and SO(2) derivatives had no significant effect on SOD activities; however, DDC and SO(2) derivatives combined led to a significant decrease of SOD activities. In the electrophysiological test, DDC (1-100mM) increased sodium current (I(Na)) in a concentration-dependent manner and the concentration for half-maximum increase (EC(50)) was 20mM. Addition of 20mM DDC to the SO(2) derivatives-containing medium significantly shifted the voltage-dependent activation curve of I(Na) toward the hyperpolarizing direction (V(h) are -51mV, -53mV and -54mV, respectively) and shifted the steady-state inactivation curve to more positive potentials (V(h) are -74mV, -71mV and -65mV, respectively) compared with the control and 10muM SO(2) derivatives exposure. These results indicated that DDC could enhance the increasing effects on Na(+) channels induced by SO(2) derivatives, and suggested that the toxicity of SO(2) on ventricular myocytes of rats was realized by free radical, especially O(2)(-).     

Role of ATP-sensitive K+ channels in electrophysiological alterations during myocardial ischemia: a study using Kir6.2-null mice

The role of cardiac ATP-sensitive K(+) (K(ATP)) channels in ischemia-induced electrophysiological alterations has not been thoroughly established. Using mice with homozygous knockout (KO) of Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene, we investigated the potential contribution of K(ATP) channels to electrophysiological alterations and extracellular K(+) accumulation during myocardial ischemia. Coronary-perfused mouse left ventricular muscles were stimulated at 5 Hz and subjected to no-flow ischemia. Transmembrane potential and extracellular K(+) concentration ([K(+)](o)) were measured by using conventional and K(+)-selective microelectrodes, respectively. In wild-type (WT) hearts, action potential duration (APD) at 90% repolarization (APD(90)) was significantly decreased by 70.1 +/- 5.2% after 10 min of ischemia (n = 6, P < 0.05). Such ischemia-induced shortening of APD(90) did not occur in Kir6.2-deficient (Kir6.2 KO) hearts. Resting membrane potential in WT and Kir6.2 KO hearts similarly decreased by 16.8 +/- 5.6 (n = 7, P < 0.05) and 15.0 +/- 1.7 (n = 6, P < 0.05) mV, respectively. The [K(+)](o) in WT hearts increased within the first 5 min of ischemia by 6.9 +/- 2.5 mM (n = 6, P < 0.05) and then reached a plateau. However, the extracellular K(+) accumulation similarly occurred in Kir6.2 KO hearts and the degree of [K(+)](o) increase was comparable to that in WT hearts (by 7.0 +/- 1.7 mM, n = 6, P < 0.05). In Kir6.2 KO hearts, time-dependent slowing of conduction was more pronounced compared with WT hearts. In conclusion, the present study using Kir6.2 KO hearts provides evidence that the activation of K(ATP) channels contributes to the shortening of APD, whereas it is not the primary cause of extracellular K(+) accumulation during early myocardial ischemia.     

Intracellular sodium in mammalian muscle fibers after eccentric contractions.

The effect of eccentric contractions on intracellular Na(+) concentration ([Na(+)](i)) and its distribution were examined in isolated rat and mouse muscle fiber bundles. [Na(+)](i) was measured with either Na(+)-binding benzofuran isophthalate or sodium green. Ten isometric contractions had no significant effect on force (measured after 5 min of recovery) and caused no significant change in the resting [Na(+)](i) (7.2 +/- 0.5 mM). In contrast 10 eccentric contractions (40% stretch at 4 muscle lengths/s) reduced developed force at 100 Hz to 45 +/- 3% of control and increased [Na(+)](i) to 16.3 +/- 1.6 mM (n = 6; P < 0.001). The rise of [Na(+)](i) occurred over 1-2 min and showed only minimal recovery after 30 min. Confocal images of the distribution of [Na(+)](i) showed a spatially uniform distribution both at rest and after eccentric contractions. Gd(3+) (20 microM) had no effect on resting [Na(+)](i) or control tetanic force but prevented the rise of [Na(+)](i) and reduced the force deficit after eccentric damage. These data suggest that Na(+) entry after eccentric contractions may occur principally through stretch-sensitive channels.     

Transmural gradients in Na/K pump activity and [Na+]I in canine ventricle

There are well-documented differences in ion channel activity and action potential shape between epicardial (EPI), midmyocardial (MID), and endocardial (ENDO) ventricular myocytes. The purpose of this study was to determine if differences exist in Na/K pump activity. The whole cell patch-clamp was used to measure Na/K pump current (I(P)) and inward background Na(+)-current (I(inb)) in cells isolated from canine left ventricle. All currents were normalized to membrane capacitance. I(P) was measured as the current blocked by a saturating concentration of dihydro-ouabain. [Na(+)](i) was measured using SBFI-AM. I(P)(ENDO) (0.34 +/- 0.04 pA/pF, n = 17) was smaller than I(P)(EPI) (0.68 +/- 0.09 pA/pF, n = 38); the ratio was 0.50 with I(P)(MID) being intermediate (0.53 +/- 0.13 pA/pF, n = 19). The dependence of I(P) on [Na(+)](i) or voltage was essentially identical in EPI and ENDO (half-maximal activation at 9-10 mM [Na(+)](i) or approximately -90 mV). Increasing [K(+)](o) from 5.4 to 15 mM caused both I(P)(ENDO) and I(P)(EPI) to increase, but the ratio remained approximately 0.5. I(inb) in EPI and ENDO were nearly identical ( approximately 0.6 pA/pF). Physiological [Na(+)](i) was lower in EPI (7 +/- 2 mM, n = 31) than ENDO (12 +/- 3 mM, n = 29), with MID being intermediate (9 +/- 3 mM, n = 22). When cells were paced at 2 Hz, [Na(+)](i) increased but the differences persisted (ENDO 14 +/- 3 mM, n = 10; EPI 9 +/- 2 mM, n = 10; and MID intermediate, 11 +/- 2 mM, n = 9). Based on these results, the larger I(P) in EPI appears to reflect a higher maximum turnover rate, which implies either a larger number of active pumps or a higher turnover rate per pump protein. The transmural gradient in [Na(+)](i) means physiological I(P) is approximately uniform across the ventricular wall, whereas transporters that utilize the transmembrane electrochemical gradient for Na(+), such as Na/Ca exchange, have a larger driving force in EPI than ENDO.     

Functionally distinct sodium channels in ventricular epicardial and endocardial cells contribute to a greater sensitivity of the epicardium to electrical depression

A greater depression of the action potential (AP) of the ventricular epicardium (Epi) versus endocardium (Endo) is readily observed in experimental models of acute ischemia and Brugada syndrome. Endo and Epi differences in transient outward K(+) current and/or ATP-sensitive K(+) channel current are believed to contribute to the differential response. The present study tested the hypothesis that the greater sensitivity of Epi is due in part to its functionally distinct early fast Na(+) current (I(Na)). APs were recorded from isolated Epi and Endo tissue slices and coronary-perfused wedge preparations before and after exposures to elevated extracellular K(+) concentration ([K(+)](o); 6-12 mM). I(Na) was recorded from Epi and Endo myocytes using whole cell patch-clamp techniques. In tissue slices, increasing [K(+)](o) to 12 mM reduced V(max) to 51.1 +/- 5.3% and 26.8 +/- 9.6% of control in Endo (n = 9) and Epi (n = 14), respectively (P < 0.05). In wedge preparations (n = 12), the increase in [K(+)](o) caused selective depression of Epi APs and transmural conduction slowing and block. I(Na) density was not significantly different between Epi (n = 14) and Endo (n = 15) cells, but Epi cells displayed a more negative half-inactivation voltage [-83.6 +/- 0.1 and -75.5 +/- 0.3 mV for Epi (n = 16) and Endo (n = 16), respectively, P < 0.05]. Our data suggest that reduced I(Na) availability in ventricular Epi may contribute to its greater sensitivity to electrical depression and thus may contribute to the R-ST segment changes observed under a variety of clinical conditions including acute myocardial ischemia, severe hyperkalemia, and Brugada syndrome.     

Apparent change in ion selectivity caused by changes in intracellular K(+) during whole-cell recording

In whole-cell recordings from HEK293 cells stably transfected with the delayed rectifier K(+) channel Kv2.1, long depolarizations produce current-dependent changes in [K(+)](i) that mimic inactivation and changes in ion selectivity. With 10 mM K(o)(+) or K(i)(+), and 140-160 mM Na(i,o)(+), long depolarizations shifted the reversal potential (V(R)) toward E(Na). However, similar shifts in V(R) were observed when Na(i,o)(+) was replaced with N-methyl-D-glucamine (NMG(+))(i, o). In that condition, [K(+)](o) did not change significantly, but the results could be quantitatively explained by changes in [K(+)](i). For example, a mean outward K(+) current of 1 nA for 2 s could decrease [K(+)](i) from 10 mM to 3 mM in a 10 pF cell. Dialysis by the recording pipette reduced but did not fully prevent changes in [K(+)](i). With 10 mM K(i,o)(+), 150 mM Na(i)(+), and 140 mM NMG(o)(+), steps to +20 mV produced a positive shift in V(R), as expected from depletion of K(i)(+), but opposite to the shift expected from a decreased K(+)/Na(+) selectivity. Long steps to V(R) caused inactivation, but no change in V(R). We conclude that current-dependent changes in [K(+)](i) need to be carefully evaluated when studying large K(+) currents in small cells.     

Differential interactions of Na+ channel toxins with T-type Ca2+ channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I(CaT) and a tetrodotoxin (TTX)-sensitive I(Ca) component (I(Ca(TTX))). In this study, we reexamined the nature of low-threshold I(Ca) in dog atrium, as well as whether it is affected by Na(+) channel toxins. Ca(2+) currents were recorded using the whole-cell patch clamp technique. In the absence of external Na(+), a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 microM TTX or micromolar concentrations of external Na(+), but was inhibited by 50 microM Ni(2+) (by approximately 90%) or 5 microM mibefradil (by approximately 50%), consistent with the reported properties of I(CaT). Addition of 30 microM TTX in the presence of Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 microM). Saxitoxin (STX) at 1 microM abolished the current left in 50 microM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 microM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 microM Ni(2+) for Ca(V)3.1 and 15 microM Ni(2+) for Ca(V)3.2); in contrast, 30 microM TTX directly inhibited hCa(V)3.3-induced I(CaT) and the addition of 750 microM Ni(2+) to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni(2+) alone. 1 microM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I(CaT) in heart, and further extend the hypothesis of a parallel evolution of Na(+) and Ca(2+) channels from an ancestor with common structural motifs.     

Intermittent hypoxia protects the rat heart against ischemia/reperfusion injury by activating protein kinase C

The aim of this study was to investigate whether and how protein kinase C (PKC) was involved in the protection afforded by intermittent hypoxia (IH) and the subcellular distribution of different PKC isozymes in rat left ventricle. Post-ischemic recovery of left ventricular developed pressure and +/-dP/dtmax in IH hearts were higher than those of normoxic hearts. Chelerythrine (CHE, 5 microM), a PKC antagonist, significantly inhibited the protective effects of IH, but had no influence on normoxic hearts. CHE significantly reduced the effect of IH on the time to maximal contracture (Tmc), but had no significant effect on the amplitude of maximal contracture (Amc) in IH group. In isolated normoxic cardiomyocytes, [Ca(2+)](i), measured as arbitrary units of fluorescence ratio (340 nm/380 nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion. However, [Ca(2+)](i) kept at normal level during simulated ischemia and reperfusion in isolated IH cardiomyocytes. In normoxic myocytes, [Na(+)](i), indicated as actual concentration undergone calibration, gradually increased during 20 min simulated ischemia and quickly declined to almost the same level as that of pre-ischemia during 30 min simulated reperfusion. However, in IH myocytes, [Na(+)](i) increased to a level lower than the corresponding of normoxic myocytes during simulated ischemia and gradually reduced to the similar level as that of normoxic myocytes after simulated reperfusion. 5 microM CHE greatly increased the levels of [Ca(2+)](i) and [Na(+)](i) during ischemia and reperfusion in normoxic and IH myocytes. In addition, we demonstrated that IH up-regulated the baseline protein expression of particulate fraction of PKC-alpha, epsilon, delta isozymes. There is no significant difference of protein expression of PKC-alpha, epsilon, delta isozymes in cytosolic fraction between IH and normoxic group. The above results suggested that PKC contributed to the cardioprotection afforded by IH against ischemia/reperfusion (I/R) injury; the basal up-regulation of the particulate fraction of PKC-alpha, epsilon, delta isozymes in IH rat hearts and the contribution of PKC to the elimination of calcium and sodium overload might underlie the mechanisms of cardioprotection by IH.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Beauvericin-induced channels in ventricular myocytes and liposomes

The antibiotic Beauvericin (BEA) was previously shown to express ionophoric properties under simple experimental systems. Its channel-forming activity was examined in inside-out patches of ventricular myocytes and synthetic membranes with the patch clamp and fluorescence imaging techniques. Current transitions to several open state levels were evident after wash-in. The BEA channel is cation-selective. Conductance and kinetics are presented for K(+) and Na(+) substates and main states. The pore was blocked by La(3+). In myocytes, the [K(+)](i) was reduced, while [Na(+)](i) and [Ca(2+)](i) increased, leading to cytolysis. These results indicate that BEA forms cation-selective channels in lipid membranes, which can affect the ionic homeostasis.     

Intracellular sodium determines frequency-dependent alterations in contractility in hypertrophied feline ventricular myocytes

Hypertrophy and failure (H/F) in humans and large mammals are characterized by a change from a positive developed force-frequency relationship (+FFR) in normal myocardium to a flattened or negative developed force-frequency relationship (-FFR) in disease. Altered Ca(2+) homeostasis underlies this process, but the role of intracellular Na(+) concentration ([Na(+)](i)) in H/F and frequency-dependent contractility reserve is unclear. We hypothesized that altered [Na(+)](i) is central to the -FFR response in H/F feline myocytes. Aortic constriction caused left ventricular hypertrophy (LVH). We found that as pacing rate was increased, contraction magnitude was maintained in isolated control myocytes (CM) but decreased in LVH myocytes (LVH-M). Quiescent LVH-M had higher [Na(+)](i) than CM (LVH-M 13.3 +/- 0.3 vs. CM 8.9 +/- 0.2 mmol/l; P < 0.001) with 0.5-Hz pacing (LVH-M 14.9 +/- 0.5 vs. CM 10.8 +/- 0.4 mmol/l; P < 0.001) but were not different at 2.5 Hz (17.0 +/- 0.7 vs. control 16.0 +/- 0.7 mmol/l; not significant). [Na(+)](i) was altered by patch pipette dialysis to define the effect of [Na(+)](i) on contraction magnitude and action potential (AP) wave shape at slow and fast pacing rates. Using AP clamp, we showed that LVH-M require increased [Na(+)](i) and long diastolic intervals to maintain normal shortening. Finally, we determined the voltage dependence of contraction for Ca(2+) current (I(Ca))-triggered and Na(+)/Ca(2+) exchanger-mediated contractions and showed that there is a greater [Na(+)](i) dependence of contractility in LVH-M. These data show that increased [Na(+)](i) is essential for maintaining contractility at slow heart rates but contributes to small contractions at fast rates unless rate-dependent AP shortening is prevented, suggesting that altered [Na(+)](i) regulation is a critical contributor to abnormal contractility in disease.     

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.     

Cardiac excitation-contraction coupling is altered in myocytes from aged male mice but not in cells from aged female mice

This study characterized age-related alterations in excitation-contraction (EC)-coupling in ventricular myocytes and investigated whether these alterations are affected by the sex of the animal. Voltage-clamp experiments were conducted in myocytes from young adult (approximately 7 mo) and aged (approximately 24 mo) male and female mice. Intracellular Ca(2+) concentrations and unloaded cell shortening were measured at 37 degrees C with fura-2 and a video edge detector. Fractional shortening and Ca(2+) current density were significantly reduced in aged male myocytes compared with those in young adult male cells. In addition, Ca(2+) transients were significantly smaller in aged male myocytes. Sarcoplasmic reticulum (SR) content, assessed by rapid application of 10 mM caffeine, declined with age in male myocytes. However, EC coupling gain and fractional release of SR Ca(2+) were similar in young adult and aged male cells. In contrast to results in male animals, fractional shortening and Ca(2+) current densities were similar in young adult and aged myocytes isolated from female hearts. Furthermore, Ca(2+) transient amplitudes were unaffected by age in female cells. Interestingly, SR Ca(2+) content was elevated in aged female myocytes, and fractional SR Ca(2+) release declined with age in females. However, the gain of EC coupling was not different in myocytes from young adult and aged female mice. These data demonstrate that age-related alterations in EC coupling are more prominent in myocytes from male hearts than in cells from female hearts and suggest that it is important to consider sex as a variable in studies of the effects of aging on cardiac EC coupling.     

Interaction of lidocaine with the cardiac sodium channel: effects of low extracellular pH are consistent with an external blocking site

Brief extracellular application of millimolar concentrations of lidocaine affected sodium currents recorded in isolated rat ventricular myocytes in two ways: 1) a reduction of the maximum current consistent with a channel blocking action, and 2) a negative shift in the voltage dependence of inactivation consistent with an interaction with the inactivated state of the channel. Both effects occurred very rapidly (< 1 s). Decreasing extracellular pH to 6.4 increased the potency for channel block (EC50 1.8 +/- 0.2 mM at pH 7.4 and 0.8 +/- 0.1 mM at pH 6.5) and decreased the potency to shift inactivation (V(1/2) shift -42 mV by 1 mM lidocaine at pH 7.4 and -12.6 mV at pH 6.5). Channel block was slightly less at +90 mV compared to -40 mV at either pH (not statistically significant). The increase in potency for block at decreased extracellular pH, while intracellular pH is buffered, and the lack of voltage dependence of block, suggest that the charged form of lidocaine can block the channel by interacting with a site near the extracellular mouth, although alternative explanations are discussed.     

K+ currents activated by depolarization in cardiac fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).     

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.     

The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.     

Effect of depolarization on binding kinetics of scorpion alpha-toxin highlights conformational changes of rat brain sodium channels.

Binding of scorpion alpha-toxins to receptor site 3 on voltage-gated sodium channels inhibits sodium current inactivation and is voltage-dependent. To reveal the direct effect of depolarization, we analyzed binding kinetics of the alpha-toxin Lqh-II (from Leiurus quinquestriatus hebraeus) to rat brain synaptosomes and effects on rat brain II (rBII) channels expressed in mammalian cells. Our results indicated that the 33-fold decrease in toxin affinity for depolarized (0 mV, 90 mM [K(+)](out), K(d) = 5.85 +/- 0.5 nM) versus polarized (-55 mV, 5 mM [K(+)](out), K(d) = 0.18 +/- 0.04 nM) synaptosomes at steady state results from a 48-fold reduction in the association rate (k(on) at 5 mM [K(+)] = (12.0 +/- 4) x 10(6) M(-1) s(-1) and (0.25 +/- 0.03) x 10(6) M(-1) s(-1) at 90 mM [K(+)](out)) with nearly no change in the dissociation rate. Electrophysiological analyses of rBII channels expressed in mammalian cells revealed that approximately 75% and 40% of rBII occupied fast- and slow-inactivated states, respectively, at resting membrane potential of synaptosomes (-55 mV), and Lqh-II markedly increased the steady-state fast and slow inactivation. To mimic electrophysiological conditions we induced fast depolarization of toxin-bound synaptosomes, which generated a biphasic unbinding of Lqh-II from toxin-receptor complexes. The first fast off rate closely resembled values determined electrophysiologically for rBII in mammalian cells. The second off rate was similar to the voltage-independent steady-state value, attributed to binding to the slow-inactivated channel states. Thus, the Lqh-II voltage-dependent affinity highlights two independent mechanisms representing conformational changes of sodium channels associated with transitions among electrically visible and invisible inactivated states.     

Altered E-C coupling in rat ventricular myocytes from failing hearts 6 wk after MI

Excitation-contraction (E-C) coupling was investigated in rat hearts 6 wk after induction of myocardial infarction (MI) by ligation of the left coronary artery. Heart weight was increased by 74% and left ventricular end-diastolic pressure was 23 +/- 2 mmHg in MI compared with 8 +/- 2 mmHg in sham-operated controls (Sham, P < 0.001). Cell shortening was measured in voltage-clamped myocytes at 36 degrees C. In solutions where Cs(+) had been replaced by K(+), the voltage dependence of contraction was sigmoidal between -20 and +100 mV in Sham and MI cells. Verapamil (20 microM) blocked L-type Ca(2+) current and reduced contraction in Sham cells by approximately 50% (P < 0.01) but did not decrease contraction significantly in MI cells at test potentials above +10 mV. Verapamil-insensitive contractions were blocked by Ni(2+) (5 mM). Na(+)/Ca(2+) exchange current was doubled in MI compared with Sham cells at test potentials between -20 and +80 mV (P < 0.05), whereas mRNA and protein expression increased by 30-40%. Finally, voltage dependence of contraction was bell shaped in Na(+)-free solutions, but contraction was significantly increased in MI cells over a wider voltage range (P < 0.05). The insensitivity to Ca(2+) channel block in MI cells may result from an increased contribution of the Na(+)/Ca(+) exchanger to triggering of E-C coupling. These results suggest significant changes in E-C coupling in the hypertrophy and failure that develop in response to extensive MI.