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).     

Effects of 8 wk of voluntary unloaded wheel running on K+ tolerance and excitability of soleus muscles in rat

During intense exercise, efflux of K(+) from working muscles increases extracellular K(+) ([K(+)](o)) to levels that can compromise muscle excitability and hence cause fatigue. In this context, the reduction in the exercise-induced elevation of [K(+)](o) observed after training in humans is suggested to contribute to the increased performance after training. Although a similar effect could be obtained by an increase in the tolerance of muscle to elevated [K(+)](o), this possibility has not been investigated. To examine this, isolated soleus muscles from sedentary (sedentary) rats and from rats that had voluntarily covered 13.1 ± 0.7 km/day in an unloaded running wheel for 8 wk (active) were compared. In muscles from active rats, the loss of force induced by exposure to an elevated [K(+)](o) of 9 mM was 42% lower than in muscles from sedentary rats (P < 0.001). This apparent increase in K(+) tolerance in active rats was associated with an increased excitability as evident from a 33% reduction in the electrical current needed to excite individual muscle fibers (P < 0.0009). Moreover, muscles from active rats had lower Cl(-) conductance, higher maximal rate of rise of single-fiber action potentials (AP), and higher Na(+)/K(+) pump content. When stimulated intermittently at 6.5 mM K(+), muscles from active rats displayed better endurance than muscles from sedentary rats, whereas no difference was found when the muscles were stimulated continuously at 30 or 120 Hz. We conclude that voluntary running increases muscle excitability, leading to improved tolerance to elevated [K(+)](o).     

Regulation of a mammalian Shaker-related potassium channel, hKv1.5, by extracellular potassium and pH

Using the whole-cell recording mode of the patch-clamp technique we studied the effects of removal of extracellular potassium, [K(+)](o), on a mammalian Shaker-related K(+) channel, hKv1.5. In the absence of [K(+)](o), current through hKv1.5 was similar to currents obtained in the presence of 4.5 mM [K(+)](o). This observation was not expected as earlier results had suggested that either positively charged residues or the presence of a nitrogen-containing residue at the external TEA(+) binding site (R487 in hKv1.5) caused current loss upon removal of [K(+)](o). However, the current loss in hKv1.5 was observed when the extracellular pH, pH(o), was reduced from 7.4 to 6.0, a behavior similar to that observed previously for current through mKv1.3 with a histidine at the equivalent position (H404). These observations suggested that the charge at R487 in hKv1.5 channels was influenced by other amino acids in the vicinity. Replacement of a histidine at position 463 in hKv1.5 by glycine confirmed this hypothesis making this H463G mutant channel sensitive to removal of [K(+)](o) even at pH(o) 7.4. We conclude that the protonation of H463 at pH 7.4 might induce a pK(a) shift of R487 that influences the effective charge at this position leading to a not fully protonated arginine. Furthermore, we assume that the charge at position 487 in hKv1.5 can directly or indirectly disturb the occupation of a K(+) binding site within the channel pore possibly by electrostatic interaction. This in turn might interfere with the concerted transition of K(+) ions resulting in a loss of K(+) conduction.     

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.     

Slow diffusion of K+ in the T tubules of rat cardiomyocytes.

Cardiomyocyte contractility is regulated by the extracellular K(+) concentration ([K(+)](o)). Potassium dynamics in the T tubules during the excitation-contraction cycle depends on the diffusion rate of K(+), but this rate is not known. Detubulation of rat cardiomyocytes was induced by osmotic shock using formamide, which separated the surface membrane from the T tubules. Changes in current and membrane potential in voltage-clamped (-80 mV) and current-clamped control and detubulated cardiomyocytes were compared during rapid switches between 5.4 and 8.1 mM [K(+)](o), and the results were simulated in a mathematical model. In the voltage-clamp experiments, the current changed significantly slower in control than in detubulated cardiomyocytes during the switch from 5.4 to 8.1 mM [K(+)](o), as indicated by the times to achieve 25, 50, 90, and 95% of the new steady-state current [control (ms) t(25) = 98 +/- 12, t(50) = 206 +/- 20, t(90) = 570 +/- 72, t(95) = 666 +/- 92; detubulated t(25) = 61 +/- 11, t(50) = 142 +/- 17, t(90) = 352 +/- 52, t(95) = 420 +/- 69]. These time points were not significantly different either during the 8.1 to 5.4 mM [K(+)](o) switch or in current-clamped cardiomyocytes switching from 5.4 to 8.1 mM [K(+)](o). Mathematical simulation of the difference current between control and detubulated cardiomyocytes gave a t-tubular diffusion rate for K(+) of approximately 85 mum(2)/s. We conclude that the diffusion of K(+) in the T tubules is so slow that they constitute a functional compartment. This might play a key role in local regulation of the action potential, and thus in the regulation of cardiomyocyte contractility.     

Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.     

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.     

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.     

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.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

Electrophysiological mechanisms of ventricular arrhythmias in relation to Andersen-Tawil syndrome under conditions of reduced IK1: a simulation study

Patients with Andersen-Tawil syndrome (ATS) mostly have mutations on the KCNJ2 gene, producing loss of function or dominant-negative suppression of the inward rectifier K(+) channel Kir2.1. However, clinical manifestations of ATS including dysmorphic features, periodic paralysis (hypo-, hyper-, or normokalemic), long QT, and ventricular arrhythmias (VAs) are considerably variable. Using a modified dynamic Luo-Rudy simulation model of cardiac ventricular myocytes, we attempted to elucidate mechanisms of VA in ATS by analyzing effects of the inward rectifier K(+) channel current (I(K1)) on the action potential (AP). During pacing at 1.0 Hz with extracellular K(+) concentration ([K(+)](o)) at 4.5 mM, a stepwise 10% reduction of Kir2.1 channel conductance progressively prolonged the terminal repolarization phase of the AP along with gradual depolarization of the resting membrane potential (RMP). At 90% reduction, early afterdepolarizations (EADs) became inducible and RMP was depolarized to -52.0 mV (control: -89.8 mV), followed by emergence of spontaneous APs. Both EADs and spontaneous APs were facilitated by a decrease in [K(+)](o) and suppressed by an increase in [K(+)](o). Simulated beta-adrenergic stimulation enhanced delayed afterdepolarizations (DADs) and could also facilitate EADs as well as spontaneous APs in the setting of low [K(+)](o) and reduced Kir2.1 channel conductance. In conclusion, the spectrum of VAs in ATS may include 1) triggered activity mediated by EADs and/or DADs and 2) abnormal automaticity manifested as spontaneous APs. These VAs can be aggravated by a decrease in [K(+)](o) and beta-adrenergic stimulation and may potentially induce torsade de pointes and cause sudden death. In patients with ATS, the hypokalemic form of periodic paralysis should have the highest propensity to VAs, especially during physical activity.     

Real time measurements of water flow in amphibian gastric glands: modulation via the extracellular Ca2+-sensing receptor

The mechanisms for the formation of the osmotic gradient driving water movements in the gastric gland and its modulation via the extracellular Ca(2+)-sensing receptor (CaR) were investigated. Real time measurements of net water flux in the lumen of single gastric glands of the intact amphibian stomach were performed using ion-selective double-barreled microelectrodes. Water movement was measured by recording changes in the concentration of impermeant TEA(+) ions ([TEA(+)](gl)) with TEA(+)-sensitive microelectrodes inserted in the lumen of individual gastric glands. Glandular K(+) (K(+)(gl)) and H(+) (pH(gl)) were also measured by using K(+)- and H(+)-sensitive microelectrodes, respectively. Stimulation with histamine significantly decreased [TEA](gl), indicating net water flow toward the gland lumen. This response was inhibited by the H(+)/K(+)-ATPase inhibitor, SCH 28080. Histamine also elicited a significant and reversible increase in [K(+)](gl) that was blocked by chromanol 293B, a blocker of KCQN1 K(+) channels. Histamine failed to induce net water flow in the presence of chromanol 293B. In the "resting state," stimulation of CaR with diverse agonists resulted in significant increase in [TEA](gl). CaR activation also significantly reduced histamine-induced water secretion and apical K(+) transport. Our data validate the strong link between histamine-stimulated acid secretion and water transport. We also show that cAMP-dependent [K(+)](gl) elevation prior to the onset of acid secretion generates the osmotic gradient initially driving water into the gastric glands and that CaR activation inhibits this process, probably through reduction of intracellular cAMP levels.     

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.     

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.     

Quaternary organic amines inhibit Na,K pump current in a voltage-dependent manner: direct evidence of an extracellular access channel in the Na,K-ATPase

The effects of organic quaternary amines, tetraethylammonium (TEA) chloride and benzyltriethylammonium (BTEA) chloride, on Na,K pump current were examined in rat cardiac myocytes superfused in extracellular Na(+)-free solutions and whole-cell voltage-clamped with patch electrodes containing a high Na(+)-salt solution. Extracellular application of these quaternary amines competitively inhibited extracellular K(+) (K(+)(o)) activation of Na,K pump current; however, the concentration for half maximal inhibition of Na,K pump current at 0 mV (K(0)(Q)) by BTEA, 4.0 +/- 0.3 mM, was much lower than the K(0)(Q) for TEA, 26.6 +/- 0.7 mM. Even so, the fraction of the membrane electric field dissipated during K(+)(o) activation of Na,K pump current (lambda(K)), 39 +/- 1%, was similar to lambda(K) determined in the presence of TEA (37 +/- 2%) and BTEA (35 +/- 2%), an indication that the membrane potential (V(M)) dependence for K(+)(o) activation of the Na,K pump current was unaffected by TEA and BTEA. TEA was found to inhibit the Na,K pump current in a V(M)-independent manner, i.e., inhibition of current dissipated 4 +/- 2% of the membrane electric field. In contrast, BTEA dissipated 40 +/- 5% of the membrane electric field during inhibition of Na,K pump current. Thus, BTEA inhibition of the Na,K-ATPase is V(M)-dependent. The competitive nature of inhibition as well as the similar fractions of the membrane electric field dissipated during K(+)(o)-dependent activation and BTEA-dependent inhibition of Na,K pump current suggest that BTEA inhibits the Na,K-ATPase at or very near the enzyme's K(+)(o) binding site(s) located in the membrane electric field. Given previous findings that organic quaternary amines are not occluded by the Na,K-ATPase, these data clearly demonstrate that an ion channel-like structure provides access to K(+)(o) binding sites in the enzyme.     

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.     

Blocking cardiac ATP-sensitive K+ channels reduces hydroxyl radicals caused by potassium chloride-induced depolarization in the rat myocardium

The current study examined whether opening of the ATP-sensitive K(+) (K(ATP)) channel can induce hydroxyl free radical (OH) generation, as detected by increases in nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) levels in the rat myocardium. When KCl (4-140mM) was administered to rat myocardium through microdialysis probe, the level of 2,3-DHBA increased gradually in a potassium ion concentration ([K(+)](o))-dependent manner. The [K(+)](o) for half-maximal effect of the level of 2,3-DHBA production (ED(50)) was 67.9microM. The maximum attainable concentration of the level of 2,3-DHBA (E(max)) was 0.171microM. Induction of glibenclamide (10microM) decreased OH formation. The half-maximal inhibitory effect (IC(50)) for glibenclamide against the [K(+)](o) (70mM)-evoked increase in 2,3-DHBA was 9.2microM. 5-Hydroxydecanoate (5-HD, 100microM), another K(ATP) channel antagonist, also decreased [K(+)](o)-induced OH formation. The IC(50) for 5-HD against the [K(+)](o) (70mM)-evoked increase in 2,3-DHBA was 107.2microM. The heart was subjected to myocardial ischemia for 15min by occlusion of left anterior descending coronary artery (LAD). When the heart was reperfused, the normal elevation of 2,3-DHBA in the heart dialysate was not observed in animals pretreated with glibenclamide (10microM) or 5-HD (100microM). These results suggest that opening of cardiac K(ATP) channels by depolarization evokes OH generation.     

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.     

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.     

Mechanisms of adrenergic control of sino-atrial node discharge

Among the mechanisms proposed for the increase in discharge of sino-atrial node (SAN) by norepinephrine (NE) are an increase in the hyperpolarization-activated current I(f) and in the slow inward current I(Ca,L). If I(f) is the primary mechanism, cesium (a blocker of I(f)) should eliminate the positive chronotropic effect of NE. If I(Ca,L), is involved, [Ca(2+)](o) should condition NE effects. We studied the electrophysiological changes induced by NE in isolated guinea pig SAN superfused in vitro with Tyrode solution (both SAN dominant and subsidiary pacemaker mechanisms are present) as well as with high [K(+)](o), higher Cs(+) or Ba(2+) (only the dominant pacemaker mechanism is present). In Tyrode solution, NE (0.5-1microM) increased the SAN rate and adding Cs(+) (approximately 12 mM) caused a decaying voltage tail during diastole in subsidiary pacemakers. NE enhanced the Cs(+)-induced tail, and increased the rate but less than in Tyrode solution. In higher [Cs(+)](o) (15- 18 mM), Ba(2+) (1 mM) or Ba(2+) plus Cs(+) (10 mM) dominant action potentials (not followed by a tail) were present and NE accelerated them as in Tyrode solution. In high [K(+)](o), NE increased the rate in the absence and presence of Cs(+), Ba(2+) or Ba(2+) plus Cs(+). In these solutions, NE increased the overshoot and maximum diastolic potential of dominant action potentials (APs) and increased the rate by steepening diastolic depolarization and shifting the threshold for upstroke to more negative values. High [Ca(2+)](o) alone increased the rate and NE enhanced this action, whereas low [Ca(2+)](o) reduced or abolished the increase in rate by NE. In SAN quiescent in high [K(+)](o) plus indapamide, NE induced spontaneous discharge by decreasing the resting potential and initiating progressively larger voltage oscillations. Thus, NE increases the SAN rate by acting primarily on dominant APs in a manner consistent with an increase of I(Ca,L) and I(K) and under conditions where I(f) is either blocked or not activated. NE INITIATES spontaneous discharge by inducing voltage oscillations unrelated to I(f).