Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Ion and voltage dependencies of the transport cycle

Ion and voltage dependencies of sodium-calcium exchange current were studied in giant membrane patches from guinea pig ventricular cells after deregulation of the exchanger with chymotrypsin. (a) Under zero-trans conditions, the half-maximum concentration (Kh) of cytoplasmic calcium (Cai) for activation of the isolated inward exchange current decreased as the extracellular sodium (Nao) concentration was decreased. The Kh of cytoplasmic sodium (Nai) for activation of the isolated outward exchange current decreased as the extracellular calcium (Cao) concentration was decreased. (b) The current-voltage (I-V) relation of the outward exchange current with saturating concentrations of Nai and Cao had a shallow slope (twofold change in approximately 100 mV) and a slight saturation tendency at very positive potentials. The outward current gained in steepness as the Nai concentration was decreased, such that the Kh for Nai decreased with depolarization. The decrease of Kh for Nai with depolarization was well described by a Boltzmann equation (e alpha.Em/26.6) with a slope (alpha) of -0.06. (c) Voltage dependence of the outward current was lost as the Cao concentration was decreased, and the Kh for Cao increased upon depolarization with a Boltzmann slope of 0.26. (d) The I-V relation of the inward exchange current, under zero-trans conditions, was also almost linear (twofold change in approximately 100 mV) and showed some saturation tendency with hyperpolarization as the Cai concentration was decreased. The Kh for Cai decreased with depolarization (Boltzmann slope, -0.10). Voltage dependence of the inward current was decreased in the presence of a high (300 mM) Nao concentration. (e) In the presence of both Na and Ca on both membrane sides, the I-V relations with saturating Nai show sigmoidal shape and clear saturation at positive potentials. Measured reversal potentials were close to the equilibrium potential expected for a 3 Na to 1 Ca exchange. (f) Nai and Cai interacted competitively with respect to the outward current, but in a mixed competitive-noncompetitive fashion with respect to the inward current. (g) Cai inhibited the outward exchange current in a voltage-dependent manner. The half-effective concentration for inhibition (Ki) by Cai increased upon depolarization with a Boltzmann slope of 0.32 in 25 mM Nai and 0.20 in 100 mM Nai. (h) Nai also inhibited the inward exchange current voltage dependently. The Ki decreased upon depolarization (Boltzmann slope, -0.11 at 3 microM Cai and -0.10 at 1.08 mM Cai).(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: reconstruction of basic cellular mechanisms

Interactions of electrogenic sodium-calcium exchange, calcium channel and sarcoplasmic reticulum in the mammalian heart have been explored by simulation of extracellular calcium transients measured with tetramethylmurexide in rabbit atrium. The approach has been to use the simplest possible formulations of these mechanisms, which together with a minimum number of additional mechanisms allow reconstruction of action potentials, intracellular calcium transients and extracellular calcium transients. A 3:1 sodium-calcium exchange stoichiometry is assumed. Calcium-channel inactivation is assumed to take place by a voltage-dependent mechanism, which is accelerated by a rise in intracellular calcium; intracellular calcium release becomes a major physiological regulator of calcium influx via calcium channels. A calcium release mechanism is assumed, which is both calcium- and voltage-sensitive, and which undergoes prolonged inactivation. 200 microM cytosolic calcium buffer is assumed. For most simulations only instantaneous potassium conductances are simulated so as to study the other mechanisms independently of time- and calcium-dependent outward current. Thus, the model reconstructs extracellular calcium transients and typical action-potential configuration changes during steady-state and non-steady-state stimulation from the mechanisms directly involved in trans-sarcolemmal calcium movements. The model predicts relatively small trans-sarcolemmal calcium movements during regular stimulation (ca. 2 mumol kg-1 fresh mass per excitation); calcium current is fully activated within 2 ms of excitation, inactivation is substantially complete within 30 ms, and sodium-calcium exchange significantly resists repolarization from approximately -30 mV. Net calcium movements many times larger are possible during non-steady-state stimulation. Long action potentials at premature excitations or after inhibition of calcium release can be supported almost exclusively by calcium current (net calcium influx 5-30 mumol kg-1 fresh mass); action potentials during potentiated post-stimulatory contractions can be supported almost exclusively by sodium-calcium exchange (net calcium efflux 4-20 mumol kg-1 fresh mass). Large calcium movements between the extracellular space and the sarcoplasmic reticulum can take place through the cytosol with virtually no contractile activation. The simulations provide integrated explanations of electrical activity, contractile function and trans-sarcolemmal calcium movements, which were outside the explanatory range of previous models.     

Cell membrane Na+, K+-ATPase and sarcoplasmic reticulum: possible regulators of intracellular ion activity.

Cardiac muscle requires an external source of calcium for contraction, but current evidence supports an intracellular pool of bound calcium as the primary activator of contraction. The size of this intracellular pool modulates the amount of calcium released to troponin during systole and the resultant contractile response. Proposed mechanisms for modulation of activator calcium include: 1) an alteration in phase II "slow current" allowing increased electrogenic calcium flux; 2) a glycoside independent sodium-calcium exchange across the sarcolemma that can be modulated by changes in the sodium gradient; 3) potassium-calcium exchange system during cardiac repolarization; 4) an augmentation of calcium accumulation by cardiac sarcoplasmic reticulum related to various phosphorylation mechanisms; and 5) an alteration in phospholipid affinity effected by cardiac glycoside at sarcolemmal sites related to the Na+, K+-ATPase.     

Na+-Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium-calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+-Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 microM), which has been shown to be a potent inhibitor of the sodium-calcium exchanger. Outward Na+-Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+-Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.     

Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Sodium-calcium exchange influences the response to endothelin-1 in lens epithelium

Studies were conducted to examine the possible involvement of Na+-Ca2+ exchanger in determining the magnitude of the endothelin-1 (ET-1)-receptor-mediated calcium signal in porcine lens epithelial cells. Cytoplasmic calcium concentration was measured in primary cultured cells loaded with Fura-2. ET-1 (100 nM) caused cytoplasmic calcium to increase transiently to approximately 250 nM from a baseline of approximately 65 nM. The calcium increase decayed to a sustained plateau 35-45 nM above the baseline. Both the peak and plateau component of the ET-1 calcium response were abolished by PD145065, an ET receptor antagonist, and by cyclopiazonic acid (CPA) (10 microM). In calcium-free bathing solution, only the plateau was abolished. In the presence of ouabain, low-sodium bathing solution or bepridil, a sodium-calcium exchange inhibitor, peak height more than doubled. Bepridil also increased the peak height of the calcium response to ATP. The half-time for decay of the ET-1 and ATP calcium peak was increased several folds by bepridil, ouabain and low-sodium conditions. Measurements of ionomycin-releasable calcium suggested calcium store size was not increased in bepridil-treated cells. Taken together findings suggest inhibition of sodium-calcium exchange increases the magnitude of the receptor-initiated store-release phase of the ET-1 calcium signaling response as the result of impaired calcium clearance from the cytoplasm.     

Effects of Taurine–Magnesium Coordination Compound on Ionic Channels in Rat Ventricular Myocytes of Arrhythmia Induced by Ouabain

Taurine-magnesium coordination compound (TMCC) has anti-arrhythmic effects. The aim of the present study was to explore the targets of the anti-arrhythmic effect of TMCC and the electrophysiological effects of TMCC on ouabain-induced arrhythmias in rat ventricular myocytes. Sodium current (I(Na)), L-type calcium current (I(ca, L)), and transient outward potassium current (I(to)) were measured and analyzed using whole-cell patch-clamp recording technique in normal rat cardiac myocytes and rat ventricular myocytes of arrhythmia induced by ouabain. In isolated ventricular myocytes, I(Na) and I(to) were blocked by TMCC (100, 200, 400 μM) in a concentration-dependent manner, and the effects of TMCC (400 μM) were equal to that of amiodarone. However, I (ca, L) was moderately increased by TMCC (400 μM) while significantly decreased by amiodarone. Ouabain (5 μM) significantly decreased sodium, L-type calcium, and transient outward potassium currents. TMCC (100 μM) relieved abnormal sodium currents induced by ouabain through facilitation of steady-state inactivation. TMCC (200 and 400 μM) relieved abnormal L-type calcium currents induced by ouabain through facilitation of steady-state activation and retardation of steady-state inactivation. TMCC failed to further inhibit abnormal transient outward potassium currents induced by ouabain. However, amiodarone inhibited the decreasing sodium, L-type calcium, and transient outward potassium currents further. These data suggest that I(Na), I(ca, L), and I(to) may be the targets of the antiarrhythmic effect of TMCC, which can antagonize ouabain-induced changes of ionic currents in rat ventricular myocytes.     

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) μ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.     

Modulation of late sodium current by Ca2+, calmodulin, and CaMKII in normal and failing dog cardiomyocytes: similarities and differences

Augmented and slowed late Na(+) current (I(NaL)) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca(2+) handling in human and canine failing myocardium. Our objective was to study I(NaL) modulation by cytosolic Ca(2+) concentration ([Ca(2+)](i)) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. I(NaL) fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na(+) channel gating, respectively. I(NaL) greatly enhanced as [Ca(2+)](i) increased from "Ca(2+) free" to 1 microM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of I(NaL) modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows I(NaL) decay and decreases the amplitude of fast exponentials, and 2) Ca(2+) shifts SSI rightward. Differences include the following: 1) slowing of I(NaL) by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca(2+) increases the amplitude of slow exponentials. We conclude that Ca(2+)/CaM/CaMKII signaling increases I(NaL) and Na(+) influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na(+) influx provides a novel Ca(2+) positive feedback mechanism (via Na(+)/Ca(2+) exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca(2+) handling in heart failure.     

Identification of Navβ1 Residues Involved in the Modulation of the Sodium Channel Nav1.4

Voltage-gated sodium channels (VGSCs) are heteromeric protein complexes that initiate action potentials in excitable cells. The voltage-gated sodium channel accessory subunit, Navβ1, allosterically modulates the α subunit pore structure upon binding. To date, the molecular determinants of the interface remain unknown. We made use of sequence, knowledge and structure-based methods to identify residues critical to the association of the α and β1 Nav1.4 subunits. The Navβ1 point mutant C43A disrupted the modulation of voltage dependence of activation and inactivation and delayed the peak current decay, the recovery from inactivation, and induced a use-dependent decay upon depolarisation at 1 Hz. The Navβ1 mutant R89A selectively delayed channel inactivation and recovery from inactivation and had no effect on voltage dependence or repetitive depolarisations. Navβ1 mutants Y32A and G33M selectively modified the half voltage of inactivation without altering the kinetics. Despite low sequence identity, highly conserved structural elements were identified. Our models were consistent with published data and may help relate pathologies associated with VGSCs to the Navβ1 subunit.     

Kinetic studies on the dissociation of adenosine cyclic 3',5'-monophosphate from the regulatory subunit of protein kinase from rabbit skeletal muscle

The exchange rate of unlabeled adenosine 3',5'-monophosphate (cAMP) with labeled [3H]cAMP in the dimeric regulatory subunit-cAMP complex of cAMP-dependent protein kinase, type I, purified from rabbit skeletal muscle is described by using the equilibrium isotope exchange technique. Results indicate that the rate of exchange carried out in the absence of the catalytic subunit (C) is rather slow with a half-life of approximately 870 s. This slow exchange rate is not affected by the presence of MgATP (50 microM). However, when both MgATP (50 microM) and C (1-13 NM) are present, the rate of isotope exchange is observed to increase markedly. Furthermore, less than stoichiometric amounts of C are required for the increase in the rate of cAMP exchange, indicating that the effect of C on the rate enhancement is a catalytic process. These results indicate that in the presence of MgATP, a ternary complex between C and regulatory subunit-cAMP complex must be formed, and a dynamic equilibrium between the eternary complex and its dissociable species must be reached within seconds. On the basis of our kinetic data, it is proposed that the formation of this ternary complex intermediate allows the rapid activation or the inactivation of cAMP-dependent protein kinase following changes in the cellular cAMP levels.     

Kinetic properties and inactivation of the gating currents of sodium channels in squid axon.

Associated with the opening and closing of the sodium channels of nerve membrane is a small component of capacitative current, the gating current. After termination of a depolarizing step the gating current and sodium current decay with similar time courses. Both currents decay more rapidly at relatively negative membrane voltages than at positive ones. The gating current that flows during a depolarizing step is diminished by a pre-pulse that inactivates the sodium permeability. A pre-pulse has no effect after inactivation has been destroyed by internal perfusion with the proteolytic enzyme pronase. Gating charge (considered as positive charge) moves outward during a positive voltage step, with voltage dependent kinetics. The time constant of the outward gating current is a maximum at about minus 10 mV, and has a smaller value at voltages either more positive or negative than this value.     

Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.     

Na+/Ca2+ exchange in cardiac myocytes. Effect of ouabain on voltage dependence.

Sarcolemmal sodium/calcium exchange activity was examined in individual chick embryonic myocardial cell aggregates that were loaded with quin 2. The baseline [Ca2+]i was 68 +/- 4 nM (n = 29). Abrupt superfusion with sodium-free lithium solution produced a fourfold increase in steady-state [Ca2+]i to 290 +/- 19 nM, which was reversible upon sodium restitution. Other methods of increasing [Ca2+]i such as KCl-depolarization or caffeine produced a dose-dependent increase in quin 2 fluorescence, accompanied by sustained contracture. The [Ca2+]i increase in zero sodium was linear, and its half-time (t1/2) of 15.1 +/- 0.1 s was similar to that of the sodium-free contracture (t1/2 = 14.4 +/- 0.5 s) under the same conditions. The sodium-dependent [Ca2+]i increase was not significantly greater when potassium served as the sodium substitute instead of lithium. This suggests that sodium/calcium exchange has little voltage dependence in this situation. However, in aggregates pretreated with ouabain (2.5 microM), the [Ca2+]i increase was almost threefold greater with potassium than with lithium (P less than 0.007). Ouabain therefore potentiated the effect of membrane potential on calcium influx. We propose that elevation of [Na2+]i is a prerequisite for voltage dependence of the sodium/calcium exchange under the conditions studied. Sodium loading will then drastically increase calcium influx during the action potential while inducing an outward membrane current that could accelerate repolarization.     

Calcium movements during each heart beat

Transsarcolemmal calcium movements are closely related to force generation in the heart. It is important to understand the transport pathways that control these movements of calcium across the sarcolemmal membrane. In the normal, beating heart, sodium-calcium exchange appears to be an important mechanism for the extrusion of calcium from the cell. The kinetics of this exchange are dependent upon the characteristics of the cell action potential. Calcium efflux via sodium-calcium exchange may be sufficient to balance calcium entry through calcium channels during the action potential.     

Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Ionic currents in two strains of rat anterior pituitary tumor cells

The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

Frequency-dependent regulation of cardiac Na(+)/Ca(2+) exchanger

The activity of the cardiac Na(+)/Ca(2+) exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na(+) and Ca(2+). In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na(+) and Ca(2+). In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na(+) and Ca(+) (Na(i)(+) and Ca(i)(2+)). Na(+)/Ca(2+) exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na(+)- and Ca(2+)-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na(+)/Ca(2+) exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca(2+), or exposure of patches to limited proteolysis by alpha-chymotrypsin, reveal that this "coupling" is due to Na(+)-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na(i)(+)-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na(+)/Ca(2+) exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.