Sodium and Calcium Inward Currents in Freshly Dissociated Smooth Myocytes of Rat Uterus

Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both I_Na and I_Ca that are vastly different from those in tissue-cultured material. I_Na is abolished by Na⁺-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na⁺ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. I_Na reactivates with a τ of 20 ms. I_Ca is abolished by Ca²⁺-free medium, Co²⁺ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca²⁺, Ba²⁺, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca²⁺ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. I_Ca reactivates with τ''s of 27 and 374 ms. I_Na and I_Ca are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak I_Na/I_Ca changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of I_Na, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with I_Na vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of I_Na, and increased densities of I_Na and I_Ca as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.     

Development and Function of the Voltage-Gated Sodium Current in Immature Mammalian Cochlear Inner Hair Cells

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca²⁺ action potentials before the onset of hearing. Although this firing activity is mainly sustained by a depolarizing L-type (Ca_V1.3) Ca²⁺ current (I _Ca), IHCs also transiently express a large Na⁺ current (I _Na). We aimed to investigate the specific contribution of I _Na to the action potentials, the nature of the channels carrying the current and whether the biophysical properties of I _Na differ between low- and high-frequency IHCs. We show that I _Na is highly temperature-dependent and activates at around −60 mV, close to the action potential threshold. Its size was larger in apical than in basal IHCs and between 5% and 20% should be available at around the resting membrane potential (−55 mV/−60 mV). However, in vivo the availability of I _Na could potentially increase to >60% during inhibitory postsynaptic potential activity, which transiently hyperpolarize IHCs down to as far as −70 mV. When IHCs were held at −60 mV and I _Na elicited using a simulated action potential as a voltage command, we found that I _Na contributed to the subthreshold depolarization and upstroke of an action potential. We also found that I _Na is likely to be carried by the TTX-sensitive channel subunits Na_V1.1 and Na_V1.6 in both apical and basal IHCs. The results provide insight into how the biophysical properties of I _Na in mammalian cochlear IHCs could contribute to the spontaneous physiological activity during cochlear maturation in vivo.     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Inhibition of L-type Ca<Superscript>2+</Superscript> current in guinea pig ventricular myocytes by cisapride

The effect of cisapride on L-type Ca²⁺ current (I _Ca,L) was studied in guinea pig ventricular myocytes using a whole-cell voltage-clamp technique and a conventional action potential recording method. Myocytes were held at –40 mV, and internally dialyzed and externally perfused with Na⁺- and K⁺-free solutions; cisapride elicited a concentration-dependent block of peakI _Ca,L, with a half-maximum inhibition concentration (IC₅₀) of 46.9 µM. There was no shift in the reversal potential, nor any change in the shape of the current-voltage relationship ofI _Ca,L in the presence of cisapride. Inhibition of cisapride was not associated with its binding to serotonin or to -adrenergic receptors because ketanserin, SB203186, and prazosin had no effect on the inhibitory action of cisapride onI _Ca,L. Cisapride elicited a tonic block and a use-dependent block ofI _Ca,L. These blocking effects were voltage dependent as the degree of inhibition at –40 mV was greater than that at –70 mV. Cisapride shifted the steady-state inactivation curve ofI _Ca,L in the negative direction, but had no effect on the steady-state activation curve. Cisapride also delayed the kinetics of recovery ofI _Ca,L from inactivation. At a slow stimulation frequency (0.1 Hz), the action potential duration in guinea pig papillary muscles showed biphasic effects; it was prolonged by lower concentrations of cisapride, but shortened by higher concentrations. These findings suggest that cisapride preferentially binds to the inactivated state of L-type Ca²⁺ channels. The inhibitory effect of cisapride onI _Ca,L might play an important role in its cardiotoxicity under pathophysiological conditions, such as myocardial ischemia.     

Distinct Ca<Superscript>2+</Superscript>-permeable Cation Currents Are Activated by Internal Ca<Superscript>2+</Superscript>-Store Depletion in RBL-2H3 Cells and Human Salivary Gland Cells,HSG and HSY

Store-operated Ca²⁺ influx, suggested to be mediated via store-operated cation channel (SOC), is present in all cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents (I _SOC) in human submandibular glands cells (HSG) and human parotid gland cells (HSY) with I _CRAC (Ca²⁺ release-activated Ca²⁺ current) in RBL cells. Internal Ca²⁺ store-depletion with IP₃ or thapsigargin activated cation channels in all three cell types. 1 μM Gd³⁺ blocked channel activity in all cells. Washout of Gd³⁺ induced partial recovery in HSY and HSG but not RBL cells. 2-APB reversibly inhibited the channels in all cells. I _CRAC in RBL cells displayed strong inward rectification with E _rev(Ca) = >+90 mV and E _rev (Na) = +60 mV. I _SOC in HSG cells showed weaker rectification with E _rev(Ca) = +25 mV and E _rev(Na) = +10 mV. HSY cells displayed a linear current with E _rev = +5 mV, which was similar in Ca²⁺- or Na⁺-containing medium. pCa/pNa was >500, 40, and 4.6 while pCs /pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY cells, respectively. Evidence for anomalous mole fraction behavior of Ca²⁺/Na⁺ permeation was obtained with RBL and HSG cells but not HSY cells. Additionally, channel inactivation with Ca²⁺ + Na⁺ or Na⁺ in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.This revised version was published online in June 2005 with a corrected cover date.     

Slow low-threshold potassium current inHelix pomatia neurons

A low-threshold outward current was studied in the neurons ofHelix pomatia at –70 to –30 mV using a two-electrode voltage clamp technique. In addition to the well-known A current (I _A), a slower outward current calledI _As (slow) was revealed. Activation and inactivation times ofI _As at –40 mV ranged from 90 to 120 msec and from 3 to 5 sec, respectively. The current recovered within 2 to 5 sec after inactivation at –120 mV. Analysis of changes in the reversal potential ofI _As caused by an increase in external potassium concentration suggests a potassium origin forI _As. The curves ofI _As stationary activation and inactivation fit the Boltzmann equation. Deriving from an activation curve, the activation potential for a half-maximum current,, is –40 mV, and the slope factor,k, is –9.8 mV, while those values for the inactivation curve are –84 mV (a half-maximum inactivation) and 7.5 mV.I _As is blocked by 4-aminopyridine (1–30 µM), tetraethylammonium (1 mM), and Ba²⁺ (1 mM), but is resistant to Cs⁺ (1 mM). PeakI _As is not affected either by substitution of external Ca²⁺ for Mg²⁺ or by application of Cd²⁺ (0.5–1.0 mM). The results suggest that activation ofI _As does not require Ca²⁺ entry into the cell.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 427–432, November–December, 1993.     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Resveratrol Attenuates the Na+-Dependent Intracellular Ca2+ Overload by Inhibiting H2O2-Induced Increase in Late Sodium Current in Ventricular Myocytes

Background/Aims

Resveratrol has been demonstrated to be protective in the cardiovascular system. The aim of this study was to assess the effects of resveratrol on hydrogen peroxide (H₂O₂)-induced increase in late sodium current (I _Na.L) which augmented the reverse Na⁺-Ca²⁺ exchanger current (I _NCX), and the diastolic intracellular Ca²⁺ concentration in ventricular myocytes.

Methods

I _Na.L, I _NCX, L-type Ca²⁺ current (I _Ca.L) and intracellular Ca²⁺ properties were determined using whole-cell patch-clamp techniques and dual-excitation fluorescence photomultiplier system (IonOptix), respectively, in rabbit ventricular myocytes.

Results

Resveratrol (10, 20, 40 and 80 µM) decreased I _Na.L in myocytes both in the absence and presence of H₂O₂ (300 µM) in a concentration dependent manner. Ranolazine (3–9 µM) and tetrodotoxin (TTX, 4 µM), I _Na.L inhibitors, decreased I _Na.L in cardiomyocytes in the presence of 300 µM H₂O₂. H₂O₂ (300 µM) increased the reverse I _NCX and this increase was significantly attenuated by either 20 µM resveratrol or 4 µM ranolazine or 4 µM TTX. In addition, 10 µM resveratrol and 2 µM TTX significantly depressed the increase by 150 µM H₂O₂ of the diastolic intracellular Ca²⁺ fura-2 fluorescence intensity (FFI), fura-fluorescence intensity change (△FFI), maximal velocity of intracellular Ca²⁺ transient rise and decay. As expected, 2 µM TTX had no effect on I _Ca.L.

Conclusion

Resveratrol protects the cardiomyocytes by inhibiting the H₂O₂-induced augmentation of I _Na.L.and may contribute to the reduction of ischemia-induced lethal arrhythmias.     

Changes in the calcium current among different transmural regions contributes to action potential heterogeneity in rat heart

To clarify the transmural heterogeneity of action potential (AP) time course, we examined the regulation of L-type Ca²⁺ current (I_Ca,L) by voltage and Ca²⁺-dependent mechanisms. Currents were recorded using patch clamp of single rat subepicardial (EPI) and subendocardial (ENDO) of left ventricular, right ventricular (RV) and septal (SEP) cardiomyocytes. Voltage clamp commands were derived from ENDO and EPI APs or rectangular voltage pulses.During rectangular pulses, peak I_Ca,L was significantly greater in EPI than in other cells. The inactivation of I_Ca,L by Ca²⁺-dependent mechanisms (suppressed by ryanodine and BAPTA) was present in all cells but greater in extent in ENDO and SEP cells. Activation and inactivation curves for all regions show subtle differences that are Ca²⁺ sensitive, with Ca²⁺ inactivation shifting the activation variables negative by ∼ 7 mV and inactivation variables positive by 2-7 mV (EPI being least, RV greatest). In AP-clamps, the peak I_Ca,L was significantly smaller in ENDO than in EPI cells, while the integrated current was significantly larger in ENDO than in EPI cells. The results are discussed with regard to the interplay of AP time course and net Ca²⁺ influx.     

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (I_A), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of −40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (I_K); and third, an outward, calcium-dependent, potassium current (I_K(Ca)) sensitive to Co²⁺ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (I_Na), blocked by tetrodotoxin, inactivated at holding potentials more positive than −40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (I_Ca,L) with a sustained time course, suppressed by nifedipine or Co²⁺, and enhanced by substituting Ca²⁺ for Ba²⁺ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current. Accepted: 24 September 1999     

Model experiments on squid axons and NG108-15 mouse neuroblastoma × rat glioma hybrid cells

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na⁺ current, the M current and the low-voltage-activated Ca²⁺ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves (Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na⁺ current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na⁺ current (PF_peak and PF_ss) as a function of voltage. Whereas PF_peak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PF_ss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na⁺ current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves (Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K⁺ current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I_M, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves (Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca²⁺ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a I_Ca. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I_Ca. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.     

Characterization of Multiple Ion Channels in Cultured Human Cardiac Fibroblasts

Background

Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts.

Methods and Findings

A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca²⁺-activated K⁺ current (BK_Ca) in most (88%) human cardiac fibroblasts, a delayed rectifier K⁺ current (IK_DR) and a transient outward K⁺ current (I_to) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K⁺ current (I_Kir) in 24% of cells, and a chloride current (I_Cl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na⁺ currents (I_Na) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (I_Na.TTX, IC₅₀ = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (I_Na.TTXR, IC₅₀ = 1.8 µM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BK_Ca), Kv1.5, Kv1.6 (responsible for IK_DR), Kv4.2, Kv4.3 (responsible for I_to), Kir2.1, Kir2.3 (for I_Kir), Clnc3 (for I_Cl), Na_V1.2, Na_V1.3, Na_V1.6, Na_V1.7 (for I_Na.TTX), and Na_V1.5 (for I_Na.TTXR).

Conclusions

These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Kinetics of the Reverse Mode of the Na<Superscript>+</Superscript>/Glucose Cotransporter

This study investigates the reverse mode of the Na⁺/glucose cotransporter (SGLT1). In giant excised inside-out membrane patches from Xenopus laevis oocytes expressing rabbit SGLT1, application of α-methyl-D-glucopyranoside (αMDG) to the cytoplasmic solution induced an outward current from cytosolic to external membrane surface. The outward current was Na⁺- and sugar-dependent, and was blocked by phlorizin, a specific inhibitor of SGLT1. The current-voltage relationship saturated at positive membrane voltages (30–50 mV), and approached zero at −150 mV. The half-maximal concentration for αMDG-evoked outward current (K_0.5^αMDG) was 35 mM (at 0 mV). In comparison, K_0.5^αMDG for forward sugar transport was 0.15 mM (at 0 mV). K_0.5^Na was similar for forward and reverse transport (≈35 mM at 0 mV). Specificity of SGLT1 for reverse transport was: αMDG (1.0) > D-galactose (0.84) > 3-O-methyl-glucose (0.55) > D-glucose (0.38), whereas for forward transport, specificity was: αMDG ≈ D-glucose ≈ D-galactose > 3-O-methyl-glucose. Thus there is an asymmetry in sugar kinetics and specificity between forward and reverse modes. Computer simulations showed that a 6-state kinetic model for SGLT1 can account for Na⁺/sugar cotransport and its voltage dependence in both the forward and reverse modes at saturating sodium concentrations. Our data indicate that under physiological conditions, the transporter is poised to accumulate sugar efficiently in the enterocyte.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.