Ionic mechanisms for the antiarrhythmic action of cinnamophilin in rat heart

We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A₂ antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (V_max) and prolonged the action potential duration at 50% repolarization (APD₅₀). Voltage clamp study showed that the suppression of V_max by Cinn was associated with an inhibition of sodium inward current (I_Na, IC₅₀=10.0 ± 0.4 µM). At 30 µM, V_1/2 for the steady-state inactivation curve of I_Na was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (I_Ca) dose-dependently with an IC₅₀ value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the I_Ca with a negative shift of V_1/2 for the steady-state inactivation curve of I_Ca from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD₅₀ was associated with an inhibition of the integral of potassium outward current with IC₅₀ values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced I_Na without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (I_to) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (K_d) of Cinn to inhibit open state I_to channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of I_to from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of I_to was calculated to be 23.9 ms. As compared with its effect on I_to, Cinn exerted about half the potency to block I_Na and I_Ca. These results indicate that the inhibition of I_Na, I_Ca and I_to may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia.     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

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.     

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.     

Nonselective Suppression of Voltage-gated Currents by Odorants in the Newt Olfactory Receptor Cells

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (V_h) of −100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na⁺ current: I_Na, T-type Ca²⁺ current: I_Ca,T, L-type Ca²⁺ current: I_Ca,L, delayed rectifier K⁺ current: I_Kv and Ca²⁺-activated K⁺ current: I_K(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (I_Na), 0.15 mM (I_Ca,T), 0.14 mM (I_Ca,L), 1.7 mM (I_Kv), and 0.17 mM (I_K(Ca)), and Hill coefficient was 1.4 (I_Na), 1.0 (I_Ca,T), 1.1 (I_Ca,L), 1.0 (I_Kv), and 1.1 (I_K(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of I_Na was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were −53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on I_Na in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by lambert-eaton syndrome IgG

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na⁺ channels are only weakly tetrodotoxin (TTX)-sensitive (K _d =215 mm). With the membrane potential maintained at –60 to –80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (>20 pA). Similar all-ornothing spikes were also observed with anodal break excitation. Removal of external Ca²⁺ did not affect the action potential production, whereas 5 m TTX or substitution of Na⁺ with choline abolished it. Action potentials elicited in the Ca²⁺-free condition were reversibly blocked by 4 mm MnCl₂ due to the Mn²⁺-induced inhibition of voltage-dependent sodium currents (I _Na). Therefore, Na⁺ channels, not Ca²⁺ channels, underlie the excitability of SCLC cells. Whole-cell I _Na was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na⁺-selective channel. I _Na evoked by depolarizing test potentials (–60 to +40 mV) exhibited a transient time course and activation/ inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m and h, also revealed biophysical similarity between SCLC and neuronal Na⁺ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at –20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit I _Ca and I _Na in bovine adrenal chromaffin cells, also significantly inhibited I _Na in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na⁺ channel conductance; (ii) fundamental characteristics of SCLC Na⁺ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na⁺ channels are an additional target of the pathological IgG present in the patients' sera.Department of Biomedical EngineeringThis study was supported by National Institutes of Health grant NS18607 and a research grant from the Muscular Dystrophy Association. Dr. Y.I. Kim is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Disorder and Stroke.     

Computational study of enhanced excitability in Hermissenda: membrane conductances modulated by 5-HT

Serotonin (5-HT) applied to the exposed but otherwise intact nervous system results in enhanced excitability of Hermissenda type-B photoreceptors. Several ion currents in the type-B photoreceptors are modulated by 5-HT, including the A-type K⁺ current (I_K,A), sustained Ca²⁺ current (I_Ca,S), Ca-dependent K⁺ current (I_K,Ca), and a hyperpolarization-activated inward rectifier current (I_h). In this study, we developed a computational model that reproduces physiological characteristics of type B photoreceptors, e.g. resting membrane potential, dark-adapted spike activity, spike width, and the amplitude difference between somatic and axonal spikes. We then used the model to investigate the contribution of different ion currents modulated by 5-HT to the magnitudes of enhanced excitability produced by 5-HT. Ion currents were systematically varied within limits observed experimentally, both individually and in combinations. A reduction of I_K,A or I_K,Ca, or an increase in I_h enhanced excitability by 20–50%. Decreasing I_Ca,S produced a dramatic decrease in excitability. Reductions of I_K,V produced only minimal increases in excitability, suggesting that I_K,V probably plays a minor role in 5-HT induced enhanced excitability. Combinations of changes in I_K,A, I_K,Ca, I_h and I_Ca,S produced increases in excitability comparable to experimental observations. After 5-HT application, the cell's depolarization force is shifted from the I_h–I_Ca,S combination to predominantly I_h.     

A prolonged,voltage-dependent calcium permeability revealed by tetraethylammonium in the soma and axon of Aplysia giant neuron

The soma but not the axon of the giant neuron, R2, of Aplysia can generate an all-or-none Ca spike in Na-free or TTX-containing medium (Junge and Miller, 1974). Extracellular axonal recordings made at several distances from the soma provide evidence that the transition in ability to fire a spike in Na-free medium occurs within the first 250 μm of the axon. Application of 25 mM TEA-Br to the bathing medium causes a more than tenfold increase in the duration of the somatic action potential. The duration of the axonal action potential in TEA decreases with distance from the soma. At distances greater than 3 mm from the soma this concentration of TEA causes little or no increase in the duration of the axon spike. The effect of 25 mM TEA on both the soma and proximal axon is blocked reversibly by 30 mM CoCl₂ or 1 mM CdCl₂. The duration of the somatic action potential in TEA increases with an increase in Ca concentration of the bath. At a constant concentration of Na, the voltage level of the somatic plateau increases with Ca concentration in the manner predicted for a Ca electrode. In the presence of 11 mM Ca²⁺ the potential of the plateau is relatively insensitive to Na concentration. The TEA plateau in R2 reveals a prolonged voltage-dependent permeability to Ca. The duration of the plateau may indicate the degree of Ca activation during a spike.     

Calcium-dependent increase in spike duration during repetitive firing of Aplysia axon in the presence of TEA

Repetitive stimulation was studied in the axon of the giant neuron, R2, of Aplysia in the presence of TEA. In 25 or 50 mM extracellular TEA, a plateau develops on the axon spike during repetitive stimulation at frequencies greater than 3/sec. The plateau in extracellular TEA is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂, and is enhanced by raising the Ca concentration. Intracellular TEA induces a plateau on the axon spike at frequencies less than 1/30sec. This plateau increases in duration with repetitive stimulation at higher frequencies and is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂. The increase in spike duration during repetitive firing in the presence of TEA is indicative of an increased entry of Ca during the spike train.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca²⁺ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K⁺-, Na⁺-free pipette solution, and bathed with K⁺-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca²⁺ current. Trials with depolarized holding potential, Ca²⁺ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na⁺ current (I_Na). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late I_Na may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I_Na.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.     

Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain

Abstract: [³H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCI) and low (200 mM KCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca²⁺, Mg²⁺, β,γ-methylene-adenosine 5′-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent K_D (nM) and B_max (fmol/mg of protein) values of 2.4 and 34, respectively, for the high-affinity site and 19.9 and 157, respectively, for the low-affinity site. Unlabeled titration/equilibrium measurements gave a single high-affinity site with a K_D value of 1.9 nM and a B_max value of 95 fmol/mg of protein. The apparent K_D value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca²⁺ (K_D/Ca²⁺= 14 nM), inhibited by Mg²⁺ (IC₆₀= 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with a K_D value similar to and a B_max value 1.8-fold greater than those obtained for the low-affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the K_D value was fivefold lower, whereas the B_max value was unaffected. The K_D value derived from association and dissociation analysis was 2.4-fold greater in 200 mM KCI compared with 1.0 M KCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg²⁺ inhibited binding was increased by 3.8-fold, whereas the affinity of the activation site for Ca²⁺ was reduced by 13-fold. Addition of caffeine in the presence of low salt increased the affinity of Ca²⁺ activation by 1.7-fold. The inhibitory effect of Mg²⁺ on [³H]-ryanodine binding in the presence of 200 mM KCI was reversed by AMP-PCP and caffeine with apparent EC₅₀ values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.     

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.     

Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.