The human etherà‐go‐go‐related gene (HERG) encodes a K+o channel that is believed to be the basis of the delayed rectified current, IKr, in cardiac muscle. We studied HERG expressed in Xenopus oocytes using a two‐electrode and cut‐open oocyte clamp technique with [K+]o of 2 and 98 mm. The time course of activation of the channel was measured using an envelope of tails protocol and demonstrated that activation of the heterologously expressed HERG current (IHERG) was sigmoidal in onset. At least three closed states were required to reproduce the sigmoid time course. The voltage dependence of the activation process and its saturation at positive voltages suggested the existence of at least one relatively voltage‐insensitive step. A three closed state activation model with a single voltage‐insensitive intermediate closed state was able to reproduce the time and voltage dependence of activation, deactivation and steady‐state activation. Activation was insensitive to changes in [K+]o. Both inactivation and recovery time constants increased with a change of [K+]o from 2 to 98 mm. Steady‐state inactivation shifted by ∼30 mV in the depolarized direction with a change from 2 to 98 mm K*o Simulations showed that modulation of inactivation is a minimal component of the increase of this current by [K+]o, and that a large increase in total conductance must also occur.

A B S TRA C T The effects of NO-related activity and cellular thiol redox state on basal L-type calcium current, ICa,L , in ferret right ventricular myocytes were studied using the patch clamp technique. SIN-I, which generates both NO-and O2-, either inhibited or stimulated ICa,L. In the presence of superoxide dismutase only inhibition was seen. 8-Br-cGMP also inhibited ICa,L, suggesting that the NO inhibition is cGMP-dependent. On the other hand, S-nitrosothiols (RSNOs), which donate NO +, stimulated ICa,L. RSNO effects were not dependent upon cell permeability, modulation of SR Ca 2+ release, activation of kinases, inhibition of phosphatases, or alterations in cGMP levels. Similar activation of Ica,L by thiol oxidants, and reversal by thiol reductants, identifies an allosteric thiol-conraining "redox switch" on the L-type calcium channel subunit complex by which NO./O 2-and NO + transfer can exert effects opposite to those produced by NO.. In sum, our results suggest that: (a) both indirect (cGMP-dependent) and direct (S-nitrosylation/oxidation) regulation of ventricular I<:a,t., and (b) sarcolemma thiol redox state may be an important determinant of ICa,L activity.KEY WORDS: cardiac electrophysiology 9 calcium homeostasis 9 ionic channels 9 N-oxides 9 S-nitrosylation

Many local anesthetics promote reduction in sodium current during repetitive stimulation of excitable membranes. Use-, frequency-, and voltage-dependent responses describe patterns of peak INa when pulse width, pulse frequency, and pulse amplitude are varied. Such responses can be viewed as reflecting voltage-sensitive shifts in equilibrium between conducting, unblocked channels and nonconducting, blocked channels. The modulated-receptor hypothesis postulates shifts in equilibrium as the result of a variable-affinity receptor and modified inactivation gate kinetics in drug-complexed channels. An alternative view considers drug blocking in the absence of these two features. We propose that drug binds to a constant-affinity channel receptor where receptor access is regulated by the channel gates. Specifically, we view channel binding sites as guarded by the channel gate conformation, so that unlike receptors where ligands have continuous access, blocking agent access is variable during the course of an action potential. During the course of an action potential, the m and h gates change conformation in response to transmembrane potential. Conducting channels with both gates open leave the binding site unguarded and thus accessible to drug, whereas nonconducting channels, with gates in the closed conformation, act to restrict drug access to unbound receptors and possibly to trap drug in drug-complexed channels. We develop analytical expressions characterizing guarded receptors as "apparently" variable-affinity binding sites and predicting shifts in "apparent" channel inactivation in the hyperpolarizing direction. These results were confirmed with computer simulations. Furthermore, these results are in quantitative agreement with recent investigations of lidocaine binding in cardiac sodium channels.

1. A fast inactivating transient K+ current (FK1) cloned from ferret ventricle and expressed in Xenopus oocytes was studied using the two-electrode voltage clamp technique. Removal of the NH2-terminal domain of FK1 (FK1A2-146) removed fast inactivation consistent with previous findings in Kv1.4 channels. The NH2-terminal deletion mutation revealed a slow inactivation process, which matches the criteria for C-type inactivation described for Shaker B channels. 2. Inactivation of FK1A2-146 at depolarized potentials was well described by a single exponential process with a voltage-insensitive time constant. In the range -90 to +20 mV, steady-state C-type inactivation was well described by a Boltzmann relationship that compares closely with inactivation measured in the presence of the NH2-terminus. These results suggest that C-type inactivation is coupled to activation. 3. The coupling of C-type inactivation to activation was assessed by mutation of the fourth positively charged residue (arginine 454) in the S4 voltage sensor to glutamine (R454Q). This mutation produced a hyperpolarizing shift in the inactivation relationship of both FK1 and FK1A2-146 without altering the rate of inactivation of either clone. 4. The rates of recovery from inactivation are nearly identical in FK1 and FK1A2-146. 5. To assess the mechanisms underlying recovery from inactivation the effects of elevated [K+]. and selective mutations in the extracellular pore and the S4 voltage sensor were compared in FK1 and FK1A2-146. The similarity in recovery rates in response to these perturbations suggests that recovery from C-type inactivation governs the overall rate of recovery of inactivated channels for both . Analysis of the rate of recovery of FK1 channels for inactivating pulses of different durations (70-2000 ms) indicates that recovery rate is insensitive to the duration of the inactivating pulse.

The biophysical characteristics and α subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21–22°C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [τrec, −70 mV = 51 ± 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [τrec, −70 mV = 3,002 ± 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 α subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 α subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito α subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4.2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito α subunits not only exists from LV epicardium to endocardium but also from apex to base.

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca 2+ and activation of Ca 2+-dependent enzymes, including nitric oxide synthase and phospholipase A 2 . Both the release of Ca 2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca 2+ from the extracellular space contribute to the [Ca 2+ ]| increase. The most important trigger for Ca 2+ release is inositol 1,4,5 -trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca 2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca 2+ channels. The magnitude of the Ca 2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, V m . Under basal conditions, V m is dominated by a large inward rectifier K + current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize V m , thus increasing the electrochemical gradient for Ca 2+ , which appears to be modulated by activation of Ca 2+ -dependent K + and CI" currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca 2+ -activated Cl~ channel) makes an estimation of their effect on endothelial cell function rather difficult. involved in the contraction of endothelial cells and the increased permeability of microvessels in response to inflammatory agents (for review, see Reference 19). Thus, a detailed knowledge of intracellular Ca 2+ homeostasis is essential for our understanding of the physiology, pathophysiology, and pharmacology of endothelial cells. The following sections review the importance of the endothelium in various disease states and the mechanisms underlying cytoplasmic Ca 2+ regulation under basal and stimulated conditions. Importance of the Endothelium in VariousDisease States Increased systemic vascular tone, which is thought to result from enhanced circulating hormones (e.g., norepinephrine, angiotensin II), is among the most common hemodynamic findings in patients with heart failure. 20 However, heightened vasoconstriction correlates poorly with the plasma levels of these substances. 20 The poor correlation could possibly be explained by the involvement of local, endothelium-dependent factors, such as an imbalance of endothelium-derived relaxing and contracting factors.21 " 23 This view is supported by numerous studies conducted in isolated vascular segments as well as in intact laboratory animals and human subjects in a variety of disease states, i...

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca 2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca 2+ ] i and NO release due to shear stress or agonist (ATP or brominated Ca 2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 μL). The microsensor was positioned ≈100 μm above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm 2 . Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca 2+ ] i , which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133±9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5±2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 μmol/L) or Br-A23187 (10 μmol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 μmol/L) chelation of extracellular Ca 2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress–induced NO release (2.23±0.07 nmol/L per second) closely resembled the kinetics of Ca 2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64±0.32 nmol/L per second). These data argue that shear stress causes a Ca 2+ -mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress. The transient nature of this response may be due to NO-induced inhibition of Ca 2+ influx via a mechanism yet to be determined.

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K + current /to were quantified. /to is selective for K +, with a PNa/P~ of 0.082. Activation of/to is a voltage-dependent process, with both activation and inactivation being independent of Na + or Ca ~+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/z = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single /to channel currents recorded in cellattached patches reproduce macroscopic/to and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22°C) ranging from 23 ms (-90 mV) to 304 ms (-1O mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22°C) are rapid, with Ito peaking in ~5-15 ms at +50 inV. Experiments conducted at reduced temperatures (12°C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22°C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism,/to gating can be described using an a 3i formulation.Address reprint requests to Dr.