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).     

NH4+-stimulated and -inhibited components of K+ transport in rice (Oryza sativa L.)

The disruption of K(+) transport and accumulation is symptomatic of NH(4)(+) toxicity in plants. In this study, the influence of K(+) supply (0.02-40 mM) and nitrogen source (10 mM NH(4)(+) or NO(3)(-)) on root plasma membrane K(+) fluxes and cytosolic K(+) pools, plant growth, and whole-plant K(+) distribution in the NH(4)(+)-tolerant plant species rice (Oryza sativa L.) was examined. Using the radiotracer (42)K(+), tissue mineral analysis, and growth data, it is shown that rice is affected by NH(4)(+) toxicity under high-affinity K(+) transport conditions. Substantial recovery of growth was seen as [K(+)](ext) was increased from 0.02 mM to 0.1 mM, and, at 1.5 mM, growth was superior on NH(4)(+). Growth recovery at these concentrations was accompanied by greater influx of K(+) into root cells, translocation of K(+) to the shoot, and tissue K(+). Elevating the K(+) supply also resulted in a significant reduction of NH(4)(+) influx, as measured by (13)N radiotracing. In the low-affinity K(+) transport range, NH(4)(+) stimulated K(+) influx relative to NO(3)(-) controls. It is concluded that rice, despite its well-known tolerance to NH(4)(+), nevertheless displays considerable growth suppression and disruption of K(+) homeostasis under this N regime at low [K(+)](ext), but displays efficient recovery from NH(4)(+) inhibition, and indeed a stimulation of K(+) acquisition, when [K(+)](ext) is increased in the presence of NH(4)(+).     

Voltage dependence of slow inactivation in Shaker potassium channels results from changes in relative K(+) and Na(+) permeabilities

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Regulation of N- and C-type inactivation of Kv1.4 by pHo and K+: evidence for transmembrane communication

Kv1.4 encodes a slowly recovering transient outward current (I(to)), which inactivates by a fast N-type (intracellular ball and chain) mechanism but has slow recovery due to C-type inactivation. C-type inactivation of the NH(2)-terminal deletion mutant (fKv1.4DeltaN) was inhibited by 98 mM extracellular K(+) concentration ([K(+)](o)), whereas N-type was unaffected. In 98 mM [K(+)](o), removal of intracellular K(+) concentration ([K(+)](i)) speeded C-type inactivation but had no effect on N-type inactivation, suggesting that C-type inactivation is sensitive to K(+) binding to intracellular sites. C-type inactivation is thought to involve closure of the extracellular pore mouth. However, a valine to alanine mutation on the intracellular side of S6 (V561A) of fKv1.4DeltaN alters recovery and results in anomalous speeding of C-type inactivation with increasing [K(+)](o). Extracellular pH (pH(o)) modulated both N- and C-type inactivation through an S5-H5 linker histidine (H508) with acidosis speeding both N- and C-type inactivation. Mutation of an extracellular lysine to a tyrosine (K532Y) slowed C-type inactivation and inhibited the pH dependence of both N- and C-type inactivation. These results suggest that mutations, [K(+)], and pH modulate inactivation through membrane-spanning mechanisms involving S6.     

A Cl(-) cotransporter selective for NH(4)(+) over K(+) in glial cells of bee retina

There appears to be a flux of ammonium (NH(4)(+)/NH(3)) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH(4)(+)/NH(3) uptake is at least partly by chloride-dependant transport of the ionic form NH(4)(+). Transmembrane transport of NH(4)(+) has been described previously on transporters on which NH(4)(+) replaces K(+), or, more rarely, Na(+) or H(+), but no transport system in animal cells has been shown to be selective for NH(4)(+) over these other ions. To see if the NH(4)(+)-Cl(-) cotransporter on bee retinal glial cells is selective for NH(4)(+) over K(+) we measured ammonium-induced changes in intracellular pH (pH(i)) in isolated bundles of glial cells using a fluorescent indicator. These changes in pH(i) result from transmembrane fluxes not only of NH(4)(+), but also of NH(3). To estimate transmembrane fluxes of NH(4)(+), it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H(+)] after its displacement with a time constant of 3 min. Membrane permeability to NH(3) was 13 microm s(-1). A numerical model was used to deduce the NH(4)(+) flux through the transporter that would account for the pH(i) changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH(4)(+)](o); the relation was fitted with a Michaelis-Menten equation with K(m) approximately 7 mM. The inhibition of NH(4)(+) flux by extracellular K(+) appeared to be competitive, with an apparent K(i) of approximately 15 mM. A simple standard model of the transport process satisfactorily described the pH(i) changes caused by various experimental manipulations when the transporter bound NH(4)(+) with greater affinity than K(+). We conclude that this transporter is functionally selective for NH(4)(+) over K(+) and that the transporter molecule probably has a greater affinity for NH(4)(+) than for K(+).     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

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.     

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.     

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.     

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.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.     

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.     

ATP from glycolysis is required for normal sodium homeostasis in resting fast-twitch rodent skeletal muscle

Myocellular sodium homeostasis is commonly disrupted during critical illness for unknown reasons. Recent data suggest that changes in intracellular sodium content and the amount of ATP provided by glycolysis are closely related. The role of glycolysis and oxidative phosphorylation in providing fuel to the Na(+)-K(+) pump was investigated in resting rat extensor digitorum longus muscles incubated at 30 degrees C for 1 h. Oxidative inhibition with carbonyl cyanide m-chlorophenylhydrazone, known as CCCP (0.2 microM), or by hypooxygenation did not alter myocellular sodium or potassium content ([Na(+)](i), [K(+)](i), respectively), whereas treatment with iodoacetic acid (0.3 mM), which effectively blocked glycolysis, dramatically increased [Na(+)](i) and the [Na(+)](i)/[K(+)](i) ratio. Experiments using ouabain and measurements of myocellular high-energy phosphates indicate that Na(+)-K(+)-ATPase activity is only impaired when glycolysis is inhibited. The data suggest that normal glycolysis is required to regulate intracellular sodium in fast-twitch skeletal muscles, because it is the predominant source of the fuel for the Na(+)-K(+)-ATPase.     

Regulation of Kv4.3 closed state inactivation and recovery by extracellular potassium and intracellular KChIP2b

Mechanisms underlying Kv4 channel inactivation and recovery are presently unclear, although there is general consensus that the basic characteristics of these processes are not consistent with Shaker (Kv1) N- and P/C-type mechanisms. Kv4 channels also differ from Shaker in that they can undergo significant inactivation from pre-activated closed-states (closed-state inactivation, CSI), and that inactivation and recovery kinetics can be regulated by intracellular KChIP2 isoforms. To gain insight into the mechanisms regulating Kv4.3 CSI and recovery, we have analyzed the effects of increasing [K(+)](o) from 2 mM to 98 mM in the absence and in the presence of KChIP2b, the major KChIP2 isoform expressed in the mammalian ventricle. In the absence of KChIP2b, high [K(+)](o) promoted Kv4.3 inactivated closed-states and significantly slowed the kinetics of recovery from both macroscopic and closed-state inactivation. Coexpression of KChIP2b in 2 mM [K(+)](o) promoted non-inactivated closed-states and accelerated the kinetics of recovery from both macroscopic and CSI. In high [K(+)](o), KChIP2b eliminated or significantly reduced the slowing effects on recovery. Attenuation of CSI by the S4 charge-deletion mutant R302A, which produced significant stabilization of non-inactivated closed-states, effectively eliminated the opposing effects of high [K(+)](o) and KChIP2b on macroscopic recovery kinetics, confirming that these results were due to alterations of CSI. Elevated [K(+)](o) therefore slows Kv4.3 recovery by stabilizing inactivated closed-states, while KChIP2b accelerates recovery by destabilizing inactivated closed-states. Our results challenge underlying assumptions of presently popular Kv4 gating models and suggest that Kv4.3 possesses novel allosteric mechanisms, which are absent in Shaker, for coupling interactions between intracellular KChIP2b binding motifs and extracellular K(+)-sensitive regulatory sites.     

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.     

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.     

Effects of calcitonin gene-related peptide on rat soleus muscle excitability: mechanisms and physiological significance

Intense exercise causes a large loss of K(+) from contracting muscles. The ensuing elevation of extracellular K(+) ([K(+)](o)) has been suggested to cause fatigue by depressing muscle fiber excitability. In isolated muscles, however, repeated contractions confer some protection against this effect of elevated K(+). We hypothesize that this excitation-induced force-recovery is related to the release of the neuropeptide calcitonin gene-related peptide (CGRP), which stimulates the muscular Na(+)-K(+) pumps. Using the specific CGRP antagonist CGRP-(8-37), we evaluated the role of CGRP in the excitation-induced force recovery and examined possible mechanisms. Intact rat soleus muscles were stimulated to evoke short tetani at regular intervals. Increasing extracellular K(+) ([K(+)](o)) from 4 to 11 mM decreased force to approximately 20% of initial force (P < 0.001). Addition of exogenous CGRP (10(-9) M), release of endogenous CGRP with capsaicin, or repeated electrical stimulation recovered force to 50-70% of initial force (P < 0.001). In all cases, force recovery could be almost completely suppressed by CGRP-(8-37). At 11 mM [K(+)](o), CGRP (10(-8) M) did not alter resting membrane potential or conductance but significantly improved action potentials (P < 0.001) and increased the proportion of excitable fibers from 32 to 70% (P < 0.001). CGRP was shown to induce substantial force recovery with only modest Na(+)-K(+) pump stimulation. We conclude that the excitation-induced force recovery is caused by a recovery of excitability, induced by local release of CGRP. The data suggest that the recovery of excitability partly was induced by Na(+)-K(+) pump stimulation and partly by altering Na(+) channel function.