The impact of age and frailty on ventricular structure and function in C57BL/6J mice.

KEY POINTS: Heart size increases with age (called hypertrophy), and its ability to contract declines. However, these reflect average changes that may not be present, or present to the same extent, in all older individuals. That aging happens at different rates is well accepted clinically. People who are aging rapidly are frail and frailty is measured with a 'frailty index'. We quantified frailty with a validated mouse frailty index tool and evaluated the impacts of age and frailty on cardiac hypertrophy and contractile dysfunction. Hypertrophy increased with age, while contractions, calcium currents and calcium transients declined; these changes were graded by frailty scores. Overall health status, quantified as frailty, may promote maladaptive changes associated with cardiac aging and facilitate the development of diseases such as heart failure. To understand age-related changes in heart structure and function, it is essential to know both chronological age and the health status of the animal. ABSTRACT: On average, cardiac hypertrophy and contractile dysfunction increase with age. Still, individuals age at different rates and their health status varies from fit to frail. We investigated the influence of frailty on age-dependent ventricular remodelling. Frailty was quantified as deficit accumulation in adult (≈7 months) and aged (≈27 months) C57BL/6J mice by adapting a validated frailty index (FI) tool. Hypertrophy and contractile function were evaluated in Langendorff-perfused hearts; cellular correlates/mechanisms were investigated in ventricular myocytes. FI scores increased with age. Mean cardiac hypertrophy increased with age, but values in the adult and aged groups overlapped. When plotted as a function of frailty, hypertrophy was graded by FI score (r = 0.67-0.55, P < 0.0003). Myocyte area also correlated positively with FI (r = 0.34, P = 0.03). Left ventricular developed pressure (LVDP) plus rates of pressure development (+dP/dt) and decay (-dP/dt) declined with age and this was graded by frailty (r = -0.51, P = 0.0007; r = -0.48, P = 0.002; r = -0.56, P = 0.0002 for LVDP, +dP/dt and -dP/dt). Smaller, slower contractions graded by FI score were also seen in ventricular myocytes. Contractile dysfunction in cardiomyocytes isolated from frail mice was attributable to parallel changes in underlying Ca2+ transients. These changes were not due to reduced sarcoplasmic reticulum stores, but were graded by smaller Ca2+ currents (r = -0.40, P = 0.008), lower gain (r = -0.37, P = 0.02) and reduced expression of Cav1.2 protein (r = -0.68, P = 0.003). These results show that cardiac hypertrophy and contractile dysfunction in naturally aging mice are graded by overall health and suggest that frailty, in addition to chronological age, can help explain heterogeneity in cardiac aging.

Cross-talk between glycogen synthase kinase 3ß (GSK3ß) and p38MAPK regulates myocyte enhancer factor 2 (MEF2) activity in skeletal and cardiac muscle.

Characterizing the signaling network that controls MEF2 transcription factors is crucial for understanding skeletal and cardiac muscle gene expression. Glycogen synthase kinase 3ß (GSK3ß) regulates MEF2 activity indirectly through reciprocal regulation of p38MAPK. Cross-talk between GSK3ß and p38MAPK regulates MEF2 activity in skeletal and cardiac muscle. Understanding cross-talk in the signaling network converging at MEF2 control has therapeutic implications in cardiac and skeletal muscle pathology. Glycogen synthase kinase 3ß (GSK3ß) is a known regulator of striated muscle gene expression suppressing both myogenesis and cardiomyocyte hypertrophy. Since myocyte enhancer factor 2 (MEF2) proteins are key transcriptional regulators in both systems, we assessed whether MEF2 is a target for GSK3ß. Pharmacological inhibition of GSK3ß resulted in enhanced MEF2A/D expression and transcriptional activity in skeletal myoblasts and cardiac myocytes. Even though in silico analysis revealed GSK3ß consensus (S/T)XXX(S/T) sites on MEF2A, a subsequent in vitro kinase assay revealed that MEF2A is only a weak substrate. However, we did observe a posttranslational modification in MEF2A in skeletal myoblasts treated with a GSK3ß inhibitor which coincided with increased p38MAPK phosphorylation, a potent MEF2A activator, indicating that GSK3ß inhibition may de-repress p38MAPK. Heart specific excision of GSK3ß in mice also resulted in up-regulation of p38MAPK activity. Interestingly, upon pharmacological p38MAPK inhibition (SB203580), GSK3ß inhibition loses its effect on MEF2 transcriptional activity suggesting potent cross-talk between the two pathways. Thus we have documented that cross-talk between p38MAPK and GSK3ß signaling converges on MEF2 activity having potential consequences for therapeutic modulation of cardiac and skeletal muscle gene expression.

Local anesthetic anchoring to cardiac sodium channels. Implications into tissue-selective drug targeting.

Local anesthetics inhibit Na+ channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na+ channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na+ channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na+ channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na+ channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.

Increased uncoupling protein-2 levels in beta-cells are associated with impaired glucose-stimulated insulin secretion: mechanism of action.

In pancreatic beta-cells, glucose metabolism signals insulin secretion by altering the cellular array of messenger molecules. ATP is particularly important, given its role in regulating cation channel activity, exocytosis, and events dependent upon its hydrolysis. Uncoupling protein (UCP)-2 is proposed to catalyze a mitochondrial inner-membrane H(+) leak that bypasses ATP synthase, thereby reducing cellular ATP content. Previously, we showed that overexpression of UCP-2 suppressed glucose-stimulated insulin secretion (GSIS) in isolated islets (1). The aim of this study was to identify downstream consequences of UCP-2 overexpression and to determine whether insufficient insulin secretion in a diabetic model was correlated with increased endogenous UCP-2 expression. In isolated islets from normal rats, the degree to which GSIS was suppressed was inversely correlated with the amount of UCP-2 expression induced. Depolarizing the islets with KCl or inhibiting ATP-dependent K(+) (K(ATP)) channels with glybenclamide elicited similar insulin secretion in control and UCP-2-overexpressing islets. The glucose-stimulated mitochondrial membrane ((m)) hyperpolarization was reduced in beta-cells overexpressing UCP-2. ATP content of UCP-2-induced islets was reduced by 50%, and there was no change in the efflux of Rb(+) at high versus low glucose concentrations, suggesting that low ATP led to reduced glucose-induced depolarization, thereby causing reduced insulin secretion. Sprague-Dawley rats fed a diet with 40% fat for 3 weeks were glucose intolerant, and in vitro insulin secretion at high glucose was only increased 8.5-fold over basal, compared with 28-fold in control rats. Islet UCP-2 mRNA expression was increased twofold. These studies provide further strong evidence that UCP-2 is an important negative regulator of beta-cell insulin secretion and demonstrate that reduced (m) and increased activity of K(ATP) channels are mechanisms by which UCP-2-mediated effects are mediated. These studies also raise the possibility that a pathological upregulation of UCP-2 expression in the prediabetic state could contribute to the loss of glucose responsiveness observed in obesity-related type 2 diabetes in humans.

P-loop flexibility in Na+ channel pores revealed by single- and double-cysteine replacements.

Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na+ channels (SkM1) caused an increased sensitivity to current blockade by Cd2+ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd2+ block relative to the individual single mutants, suggesting coordinated Cd2+ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd2+ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore.

To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+ > Li+ > NH4+ > > Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+ and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+ > K+ > Na+ > or = Li+ approximately Cs+), D1532C, and G1533C (Na+ > Li+ > or = NH4+ > K+ > Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4+ > K+ > Na+ > or = Li+ approximately Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+ > or = Sr2+ > Mg2+ > Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.

Antibodies against Tityus discrepans venom do not abolish the effect of Tityus serrulatus venom on the rat sodium and potassium channels.

Anti-(Tityus serrulatus + Tityus bahiensis) and anti-Tityus discrepans venom polyclonal antisera were used to investigate whether antigenic differences exist between the venoms of the Brazilian T. serrulatus and the Venezuelan T. discrepans scorpions. Both antisera recognised the toxin-containing electrophoretic fractions of their cognate venoms and also those from Tityus zulianus and Tityus trinitatis venoms on Western blots. The anti-T. discrepans antiserum reacted only weakly with T. serrulatus toxic polypeptides. The effect of T. serrulatus alpha- or beta-toxins on rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes was abolished by pre-incubating the venom with anti-(T. serrulatus + T. bahiensis) serum but not with anti-T. discrepans serum. Nor did the Brazilian or the Venezuelan sera prevent the reduction in K+ currents by T. serrulatus venom in X. laevis oocytes expressing the rat brain delayed rectifying Shaker K+ channel (Kv1.2). These results indicate that toxins from T. serrulatus and T. discrepans venoms, which primarily target mammalian Na+ channels, are antigenically distinct, although they probably share common epitopes. Our results also suggest that Na+ channel-active toxins are the immunodominant antigens of the T. serrulatus venom.

Nutrient responsive nesfatin-1 regulates energy balance and induces glucose-stimulated insulin secretion in rats.

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic ß-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

Concentration-dependent effects of purine/xanthine oxidase on release of 6-keto-PGF1 alpha and contractile function of isolated guinea pig hearts: response to "ischemic" conditions followed by reperfusion.

This study was designed to demonstrate the concentration-dependent effects of an exogenous free-radical-generating system on the functional characteristics of isolated perfused guinea pig hearts under normal conditions and in response to conditions associated with ischemia followed by reperfusion. Purine (0.0115-0.23 mM) and xanthine oxidase (0.05-1.0 U/L) were added to normal Tyrode's solution and perfused for 40 min. Purine (0.0575-0.23 mM)/xanthine oxidase (0.25-1.0 U/L) produced a decline in contractile force that ranged from 59 to 44% of initial values (p less than 0.05). Although all concentrations of the free-radical-generating system enhanced resting tension when compared to control, this increase was only significant in the presence of purine (0.0115 and 0.0575 mM)/xanthine oxidase (0.05 and 0.25 U/L), following a 20-40 min perfusion period (p less than 0.05). Significant correlations were found between the concentration of the free-radical-generating system and the depression in contractile force (p less than 0.05), as well as between the loss of force and the enhancement of resting tension (p less than 0.002) in the presence of all concentrations of purine/xanthine oxidase examined. Furthermore, purine/xanthine oxidase was a potent stimulus for release of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). While this release was correlated significantly with the concentration of purine/xanthine oxidase (p less than 0.001), there was no significant relationship between the decline in contractile force and the release of 6-keto-PGF1 alpha per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and electrophysiological effects of quinacrine on the response of ventricular tissues to hypoxia and reoxygenation.

This study examined the effects of quinacrine on the functional and electrophysiological responses of isolated guinea pig hearts and isolated canine papillary muscle and Purkinje fibre preparations. A dose-response relationship for quinacrine (0.01-10.0 micrograms/mL) was studied in isolated guinea pig hearts perfused for 40 min. Quinacrine was found to exert a concentration-dependent negative inotropic effect (1.0 and 10 micrograms/mL); in the presence of the 10 micrograms/mL of the drug, hearts developed contracture, atrioventricular conduction block, and ventricular asystole. In hearts exposed to hypoxia, lactate acidosis, and glucose deprivation and then reoxygenated for 30 min, pretreatment with quinacrine (0.1 microgram/mL) for 15 min prior to the initiation of hypoxia resulted in enhanced recovery of contractile function. Administration of the drug at any other time of the hypoxia-reoxygenation protocol was without effect. However, quinacrine reduced both the incidence and duration of reoxygenation arrhythmias. To examine the possible mechanistic basis for this antiarrhythmic action, isolated canine preparations were exposed to the same conditions and then reoxygenated. Quinacrine (1 microgram/mL) significantly reduced the reoxygenation-associated loss in membrane potential and prevented inexcitability and depolarization-induced automaticity in Purkinje fibres. These results suggest that quinacrine exerts an antiarrhythmic action during reoxygenation and may do so by modifying some potential mechanisms of arrhythmia that occur in the specialized conduction system.

Differential effects of purine/xanthine oxidase on the electrophysiologic characteristics of ventricular tissues.

Oxygen free radicals (OFR) exert direct effects on the electrophysiologic properties of a variety of cardiac preparations under well-oxygenated conditions and have been implicated in the genesis of arrhythmias associated with myocardial ischemia and reperfusion. In the present study, we examined the effects of the OFR generating system, purine and xanthine oxidase, on the intracellular electrical activity of canine Purkinje fibers and papillary muscles. Purine and xanthine oxidase generated a concentration-dependent production of superoxide anion. This was accompanied by a time- and concentration-dependent depolarization of the membrane potential and shortening of the action potential duration (APD50 and APD90) in Purkinje fibers. There was no significant effect of any concentration of purine/xanthine oxidase tested on these parameters in the papillary muscles. In addition, we observed a frequency-dependent change in the sensitivity of Purkinje fibers exposed to purine/xanthine oxidase both with respect to the concentration- and time-dependent effects on APD. In the presence of 5.75 mM purine and 25 U/L xanthine oxidase, APD was significantly shortened after 10 min when the Purkinje fiber was stimulated at a basic cycle length (BCL) of 300 ms. At a BCL of 500 ms, APD did not shorten significantly until 40 min. At longer BCL (greater than 800 ms), prolonged periods of exposure were required before any significant change in APD was observed. These results suggest that OFR can alter the electrophysiologic characteristics of cardiac tissues directly and that these effects could potentially exert a proarrhythmic effect, particularly under conditions in which heart rate (HR) is elevated.

Characteristics of cocaine block of purified cardiac sarcoplasmic reticulum calcium release channels.

We have examined the effects of cocaine on the SR Ca2+ release channel purified from canine cardiac muscle. Cocaine induced a flicker block of the channel from the cytoplasmic side, which resulted in an apparent reduction in the single-channel current amplitude without a marked reduction in the single-channel open probability. This block was evident only at positive holding potentials. Analysis of the block revealed that cocaine binds to a single site with an effective valence of 0.93 and an apparent dissociation constant at 0 mV (Kd(0)) of 38 mM. The kinetics of cocaine block were analyzed by amplitude distribution analysis and showed that the voltage and concentration dependence lay exclusively in the blocking reaction, whereas the unblocking reaction was independent of both voltage and concentration. Modification of the channel by ryanodine dramatically attenuated the voltage and concentration dependence of the on rates of cocaine block while diminishing the off rates to a lesser extent. In addition, ryanodine modification changed the effective valence of cocaine block to 0.52 and the Kd(0) to 110 mM, suggesting that modification of the channel results in an alteration in the binding site and its affinity for cocaine. These results suggest that cocaine block of the SR Ca2+ release channel is due to the binding at a single site within the channel pore and that modification of the channel by ryanodine leads to profound changes in the kinetics of cocaine block.

Inactivated state dependence of sodium channel modulation by beta-scorpion toxin.

We have examined the effects of a beta-scorpion toxin purified from the venom of the Venezuelan scorpion Tityus discrepans, TdVIII, on heterologously expressed rat skeletal muscle Na+ channels (rSkM1). TdVIII (100 nM) produced a leftward shift in the voltage dependence of activation and reduced the peak Na+ conductance of rSkM1 channels coexpressed with the rat brain beta1 subunit in Xenopus laevis oocytes, suggesting that TdVIII is a beta-scorpion toxin. These effects did not depend on the presence of the beta1 subunit. Modification of rSkM1 activation by TdVIII could be augmented by increasing the rate of stimulation (enhanced use-dependence). Shifts in channel activation were also enhanced by introducing conditioning pulses to -10 mV, and this enhancement increased with conditioning pulse duration. On the other hand, TdVIII did not affect the activation of fast-inactivation deficient mutant Na+ channels, I1303Q/ F1304Q/M1305Q. These results suggest that modulation of rSkM1 Na+ channel gating by TdVIII depends on the toxin interacting with the inactivated state of the alpha subunit.

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation.

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+ channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopus oocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to -10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at -80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (tauWT = 10.8 +/- 0.9 ms; taueqHPP = 2.9 +/- 0.4 ms). Benzocaine slowed the time course of recovery (taueqHPP,ben = 9.6 +/- 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (taufast,lid = 3.2 +/- 0.2 ms; taufast,mex = 3.1 +/- 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (tauslow,lid = 1,688 +/- 180 ms; tauslow,mex = 2,323 +/- 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.

Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis.

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-CTX sensitivity (IC50 = 1.9 +/- 0.1, 4.9 +/- 0.9, and 5.5 +/- 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan-to-cysteine mutants partially or fully restored the wild-type mu-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.

Effect of Cd2+ on Kv4.2 and Kv1.4 expressed in Xenopus oocytes and on the transient outward currents in rat and rabbit ventricular myocytes.

The aim of the present study was to compare the biophysical properties and Cd2+ sensitivity of Kv4.2 and Kv1.4 in Xenopus oocytes with those of native transient outward potassium currents in rat and rabbit ventricular myocytes. In Xenopus oocytes, Kv4.2 inactivated at hyperpolarized voltages (V(1/2)inact = -58.4 +/- 0.96 mV, n = 12) and recovered from inactivation rapidly (time constant = 224 +/- 23 ms, n = 3). Cd2+ induced large (approx. 30 mV with 500 microM Cd2+), concentration-dependent rightward shifts in Kv4.2 steady-state activation and inactivation. Kv1.4 inactivated over more depolarized voltages than Kv4.2 (V(1/2)inact = -49.3 +/- 1.4 mV, n = 12). Recovery from inactivation of Kv1.4 was dominated by a large slow component (time constant = 9,038 +/- 1,178 ms, n = 4). Cd2+ exerted only modest effects on Kv1.4 gating, with 500 microM Cd2+ shifting the voltage dependence of steady-state activation and inactivation by approximately 12 mV. We show that the biophysical properties and Cd2+ sensitivity of rat ventricular Ito resemble those of heterologously expressed Kv4.2. These findings support previous suggestions that Kv4.2 is an important molecular component of Ito in adult rat heart. In addition, our findings show that Ito in rabbit ventricular myocytes and Kv1.4-based currents in Xenopus oocytes share similar biophysical properties and sensitivity to Cd2+, suggesting that Kv1.4 may underlie Ito in rabbit ventricle. However, a number of discrepancies exist between the properties of native currents and their putative molecular counterparts, suggesting that additional proteins and/or modulatory factors may also play a role in determining the biophysical and pharmacological properties of these native currents.

The equine periodic paralysis Na+ channel mutation alters molecular transitions between the open and inactivated states.

1. The Na+ channel mutation associated with equine hyperkalaemic periodic paralysis (HPP) affects a highly conserved phenylalanine residue in an unexplored region of the alpha-subunit. This mutation was introduced into the rat skeletal muscle Na+ channel gene at the corresponding location (i.e. F1412L) for functional expression and characterization in Xenopus oocytes. 2. In comparison with wild-type (WT) channels, equine HPP channels showed clear evidence for disruption of inactivation: increased time-to-peak current, slowed rates of whole-cell current decay, significant increases in sustained current, rightward shifts in the steady-state inactivation curve by 9.5 mV, a 6-fold acceleration in the rate of recovery from inactivation at -80 mV, decreased number of blank single-channel sweeps, repetitive opening of single channels throughout depolarizing steps, increased open probability per sweep, and an increased mean open time. 3. The observed disruption of inactivation in HPP occurred without measurable changes in steady-state activation and first latency kinetics of channel opening. 4. Kinetic modelling demonstrates that the equine HPP phenotype can be simulated by altering the rate constants for transitions entering and leaving the inactivated states resulting from an energetic destabilization of the inactivated state. 5. These results suggest that the highly conserved cytoplasmic end of the third transmembrane segment (S3) in the fourth internal repeat domain (domain IV) plays a critical role in Na+ channel inactivation.

Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1.

Inwardly rectifying potassium channels are important in cellular repolarization of many excitable tissues. Amino acid sequence alignment of different mammalian inward rectifier K(+) channels revealed two absolutely conserved cysteine residues in the putative extracellular face, suggesting a possible disulfide bond. Replacement of these cysteine residues in the Kir2.1 channel (i.e., C122 and C154) with either alanine or serine abolished current in Xenopus laevis oocytes although Western blotting established that the channels were fully expressed. The digestion pattern of channels treated with V8 protease combined with Western blotting under reducing and nonreducing conditions confirmed intrasubunit cross-linking of C122 and C154. Whole-cell and single channel current recordings of oocytes expressing tandem tetrameric constructs with one or two of the mutant subunits suggested that insertion of one mutant subunit is sufficient to eliminate channel function. Coexpression studies confirmed that the cysteine mutant channels eliminate wild-type Kir2.1 currents in a dominant-negative manner. Despite these results, sulfhydryl reduction did not alter the functional properties of Kir2.1 currents. Molecular modeling of Kir2.1 with the two cysteines cross-linked predicted that the extracellular loop between the first transmembrane domain and the pore helix contains a beta-hairpin structure. Distinct from the KcsA structure, the disulfide bond together with the beta-hairpin structure is expected to constrain and stabilize the P-loop and selectivity filter. Taken together, these results suggest that intramolecular disulfide bond exists between C122 and C154 of Kir2.1 channel and this cross-link might be required for proper channel folding.

Modification of cardiac Na(+) current by RWJ 24517 and its enantiomers in guinea pig ventricular myocytes.

We examined the effects of the cardiotonic agent RWJ 24517 (Carsatrin, racemate) and its (S)- and (R)-enantiomers on action potential duration, Na(+) current (I(Na)), and delayed rectifier K(+) current (I(K)) of guinea pig ventricular myocytes. RWJ 24517 (0. 1 and 1 microM) prolongation of action potential duration could not be accounted for by suppression of either the rapid (I(Kr)) or slow (I(Ks),) component of I(K), although RWJ 24517 did reduce I(Kr) at concentrations of 1 microM. A more dramatic effect of RWJ 24517 (0.1-1 microM) and the (S)-enantiomer of RWJ 24517 (0.1-3 microM) was an increase in peak I(Na) and slowing of the rate of I(Na) decay, eliciting a large steady-state current. Neither RWJ 24517 nor the (S)-enantiomer affected the fast time constant for I(Na) decay, but both significantly increased the slow time constant, in addition to increasing the proportion of I(Na) decaying at the slow rate. Both agents elicited a use-dependent decrease of peak I(Na) (3-10 microM), which probably resulted from a slowing of both fast and slow rates of recovery from inactivation. In contrast, the (R)-enantiomer of RWJ 24517 did not induce a steady-state component I(Na) or increase peak I(Na) up to 10 microM, but it decreased peak I(Na) at 30 microM. The (R)-enantiomer displayed little use-dependent reduction of I(Na) during trains of repetitive pulses and had no effect on rates of inactivation or recovery from inactivation. These actions of the racemate and the (S)-stereoisomer to slow inactivation and to prolong both Na(+) influx and action potential duration may contribute to the positive inotropic actions of these agents because the resulting accumulation of intracellular Na(+) would increase intracellular Ca(2+) via Na(+)/Ca(2+) exchange.

Preferential regulation of rabbit cardiac L-type Ca2+ current by glycolytic derived ATP via a direct allosteric pathway.

1. The activity of Ca2+ channels is regulated by a number of mechanisms including direct allosteric modulation by intracellular ATP. Since ATP derived from glycolysis is preferentially used for membrane function, we hypothesized that glycolytic ATP also preferentially regulates cardiac L-type Ca2+ channels. 2. To test this hypothesis, peak L-type Ca2+ currents (ICa) were measured in voltage-clamped rabbit cardiomyocytes during glycolytic inhibition (2-deoxyglucose + pyruvate), oxidative inhibition (cyanide + glucose) or both (full metabolic inhibition; FMI). 3. A 10 min period of FMI resulted in a 40.0 % decrease in peak ICa at +10 mV (-5.1 +/- 0.6 versus -3.1 +/- 0.4 pA pF-1; n = 5, P < 0.01). Similar decreases in peak ICa were observed during glycolytic inhibition using 2-deoxyglucose (-6.2 +/- 0.2 versus -3.7 +/- 0.2 pA pF-1; n = 5, P < 0.01) or iodoacetamide (-6.7 +/- 0.3 versus -3.7 +/- 0.2 pA pF-1; n = 7, P < 0.01), but not following oxidative inhibition (-6.2 +/- 0.4 versus -6.4 +/- 0.3 pA pF-1; n = 5, n.s.). The reduction in ICa following glycolytic inhibition was not mediated by phosphate sequestration by 2-deoxyglucose or changes in intracellular pH. 4. Reductions in ICa were still observed when inorganic phosphate and creatine were included in the pipette, confirming a critical role for glycolysis in ICa regulation. 5. With 5 mM MgATP in the pipette during FMI, peak ICa decreased by only 18.4 % (-6.8 +/- 0.6 versus -5.5 +/- 0.3 pA pF-1; n = 4, P < 0.05), while inclusion of 5 mM MgAMP-PCP (beta,gamma-methyleneadenosine 5'-triphosphate, Mg2+ salt) completely prevented the decrease in peak ICa (-6.9 +/- 0.3 versus -6.5 +/- 0.3 pA pF-1; n = 5, n.s.). 6. Together, these results suggest that ICa is regulated by intracellular ATP derived from glycolysis and does not require hydrolysis of ATP. This regulation is expected to be energy conserving during periods of metabolic stress and myocardial ischaemia.