A model of the guinea-pig ventricular cardiac myocyte incorporating a transverse-axial tubular system

A model of the guinea-pig cardiac ventricular myocyte has been developed that includes a representation of the transverse–axial tubular system (TATS), including heterogeneous distribution of ion flux pathways between the surface and tubular membranes. The model reproduces frequency-dependent changes of action potential shape and intracellular ion concentrations and can replicate experimental data showing ion diffusion between the tubular lumen and external solution in guinea-pig myocytes. The model is stable at rest and during activity and returns to rested state after perturbation. Theoretical analysis and model simulations show that, due to tight electrical coupling, tubular and surface membranes behave as a homogeneous whole during voltage and current clamp (maximum difference 0.9 mV at peak tubular I_Na of −38 nA). However, during action potentials, restricted diffusion and ionic currents in TATS cause depletion of tubular Ca²⁺ and accumulation of tubular K⁺ (up to −19.8% and +3.4%, respectively, of bulk extracellular values, at 6 Hz). These changes, in turn, decrease ion fluxes across the TATS membrane and decrease sarcoplasmic reticulum (SR) Ca²⁺ load. Thus, the TATS plays a potentially important role in modulating the function of guinea-pig ventricular myocyte in physiological conditions.     

Effects of sustained beta-adrenergic stimulation on ionic currents of cultured adult guinea pig cardiomyocytes

Short-term stimulation of beta-receptors is known to affect cardiac ion channels; however, the impact of longer-term stimulation on intrinsic channel function is poorly understood. To evaluate this, cultured guinea pig ventricular myocytes were exposed to isoproterenol (10 nM), vehicle, or isoproterenol plus propranolol (1 microM) for 48 h. Sustained exposure to isoproterenol decreased the density of the inward rectifier (I(K1)), slow delayed rectifier (I(Ks)), and L-type Ca2+ (I(Ca L)) currents, effects that were fully prevented by propranolol. Changes in K+ currents were prevented by the beta1-selective antagonist CGP-20712A, unaffected by the beta2-antagonist ICI-118,551, and mimicked by the membrane-permeable cAMP analog 8-bromo-cAMP. Isoproterenol did not alter the current-voltage relationship of the K+ currents but increased the density of T-type Ca2+ current (I(Ca T)) and thereby increased the proportion of the total Ca2+ current at more negative potentials. We conclude that sustained exposure to isoproterenol reduces I(K1), I(Ks), and I(Ca L) density and increases the density of I(Ca T). The direct ionic current remodeling effects of sustained beta-adrenoceptor stimulation resemble changes reported with heart failure and may be important in arrhythmogenic ionic remodeling.     

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.     

Time-dependent transients in an ionically based mathematical model of the canine atrial action potential

Ionically based cardiac action potential (AP) models are based on equations with singular Jacobians and display time-dependent AP and ionic changes (transients), which may be due to this mathematical limitation. The present study evaluated transients during long-term simulated activity in a mathematical model of the canine atrial AP. Stimulus current assignment to a specific ionic species contributed to stability. Ionic concentrations were least disturbed with the K(+) stimulus current. All parameters stabilized within 6-7 h. Inward rectifier, Na(+)/Ca(2+) exchanger, L-type Ca(2+), and Na(+)-Cl(-) cotransporter currents made the greatest contributions to stabilization of intracellular [K(+)], [Na(+)], [Ca(2+)], and [Cl(-)], respectively. Time-dependent AP shortening was largely due to the outward shift of Na(+)/Ca(2+) exchange related to intracellular Na(+) (Na) accumulation. AP duration (APD) reached a steady state after approximately 40 min. AP transients also occurred in canine atrial preparations, with the APD decreasing by approximately 10 ms over 35 min, compared with approximately 27 ms in the model. We conclude that model APD and ionic transients stabilize with the appropriate stimulus current assignment and that the mathematical limitation of equation singularity does not preclude meaningful long-term simulations. The model agrees qualitatively with experimental observations, but quantitative discrepancies highlight limitations of long-term model simulations.     

Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

Surface potentials measure ion concentrations near lipid bilayers during rapid solution changes.

We describe a puffing method for changing solutions near one surface of lipid bilayers that allows simultaneous measurement of channel activity and extent of solution change at the bilayer surface. Ion adsorption to the lipid headgroups and screening of the bilayer surface charge by mobile ions provided a convenient probe for the ionic composition of the solution at the bilayer surface. Rapid ionic changes induced a shift in bilayer surface potential that generated a capacitive transient current under voltage-clamp conditions. This depended on the ion species and bilayer composition and was accurately described by the Stern-Gouy-Chapman theory. The time course of solute concentrations during solution changes could also be modeled by an exponential exchange of bath and puffing solutions with time constants ranging from 20 to 110 ms depending on the flow pressure. During changes in [Cs+] and [Ca2+] (applied separately or together) both the mixing model and capacitive currents predicted [Cs+] and [Ca2+] transients consistent with those determined experimentally from: 1) the known Cs(+)-dependent conductance of open ryanodine receptor channels and 2) the Ca(2+)-dependent gating of ryanodine receptor Ca2+ channels from cardiac and skeletal muscle.     

Kinetic and steady-state properties of Na+ channel and Ca2+ channel charge movements in ventricular myocytes of embryonic chick heart

Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.     

Ionic currents of channels that are permeable to monovalent and divalent cations.

The cation-selective channel from Tetrahymena cilia is permeable to both monovalent and divalent cations. The single channel conductance in mixed solutions of K+ and Ca2+ was determined by the Gibbs-Donnan ratio of K+ and Ca2+, and the binding sites of this channel were considered to be always occupied by two potassium ions or by one calcium ion under the experimental conditions: 5-90 mM K+ and 0.5-35 mM Ca2+ (Oosawa and Kasai, 1988). A two-barrier model for the channel was introduced and the values of Michaelis-Menten constants and maximum currents carried by K+ and Ca2+ were calculated using this model. Single channel current amplitudes and reversal potentials were calculated from these values. The calculated single-channel currents were compared with those obtained experimentally. The calculated reversal potentials were compared with the resting potentials of Tetrahymena measured in various concentrations of extracellular K+ and Ca2+. The method of calculation of ionic currents and reversal potentials presented here is helpful for understanding the properties of the channels permeable to both monovalent and divalent cations.     

Frequency-dependent and proarrhythmogenic effects of FK-506 in rat ventricular cells

FK-506, a widely used immunosuppressant, has caused a few clinical cases with QT prolongation and torsades de pointe at high blood concentration. The proarrhytmogenic potential of FK-506 was investigated in single rat ventricular cells using the whole cell clamp method to record action potentials (APs) and ionic currents. Fluorescence measurements of Ca2+ transients were performed with indo-1 AM using a multiphotonic microscope. FK-506 (25 micromol/l) hyperpolarized the resting membrane potential (RMP; -3 mV) and prolonged APs (AP duration at 90% repolarization increased by 21%) at 0.1 Hz. Prolongation was enhanced by threefold at 3.3 Hz, and early afterdepolarizations (EADs) occurred in 59% of cells. EADs were prevented by stronger intracellular Ca2+ buffering (EGTA: 10 vs. 0.5 mmol/l in the patch pipette) or replacement of extracellular Na+ by Li+, which abolishes Na+/Ca2+ exchange [Na+/Ca2+ exchanger current (INaCa)]. In indo-1-loaded cells, FK-506 generated doublets of Ca(2+) transients associated with increased diastolic Ca2+ in one-half of the cells. FK-506 reversibly decreased the L-type Ca2+ current (ICaL) by 25%, although high-frequency-dependent facilitation of ICaL persisted, and decreased three distinct K+ currents: delayed rectifier K+ current (IK; >80%), transient outward K+ current (<20%), and inward rectifier K+ current (IK1; >40%). A shift in the reversal potential of IK1 (-5 mV) accounted for RMP hyperpolarization. Numerical simulations, reproducing all experimental effects of FK-506, and the use of nifedipine showed that frequency-dependent facilitation of ICaL plays a role in the occurrence of EADs. In conclusion, the effects of FK-506 on the cardiac AP are more complex than previously reported and include inhibitions of IK1 and ICaL. Alterations in Ca2+ release and INaCa may contribute to FK-506-induced AP prolongation and EADs in addition to the permissive role of ICaL facilitation at high rates of stimulation.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

Calcineurin,a Type 2B Protein Phosphatase,Modulates the Ca2+-Permeable Slow Vacuolar Ion Channel of Stomatal Guard Cells

The slowly activating vacuolar (SV) channel of plant vacuoles is gated open by cytosolic free Ca2+ and by cytosol-positive potentials. Using vacuoles isolated from broad bean guard cell protoplasts, SV-mediated currents could be measured in the whole-vacuole configuration of a patch clamp as the time-dependent increase in current at cytosol-positive voltages. Time-dependent deactivation of the SV currents when changing from activating to nonactivating voltages (tail currents) was used to calculate the selectivity of the channel to Ca2+ and Cl- with respect to K+. Changing the equilibrium potential for each permeant ion (Ca2+, Cl-, and K+) at least once for individual vacuoles allowed the relative permeabilities (P) of each of these ions to be calculated in a single experiment. The resulting Pca:Pcl:Pk ratio was close to 3:0.1:1. In accord with its characterization as a weakly selective Ca2+ channel, the SV-mediated current density decreased with increasing Ca2+ activity in the vacuole lumen. SV currents were potently modulated by the Ca2+-dependent, calmodulin-stimulated protein phosphatase 2B (calcineurin). At low concentrations ([less than or equal to]0.4 units per mL), calcineurin stimulated SV currents by ~60%, whereas at higher concentrations the phosphatase was inhibitory, reaching ~90% inhibition at 3 units per mL. Bovine calmodulin had no direct effect on SV-mediated currents, although calcineurin stimulated by exogenous calmodulin inhibited SV currents at all concentrations tested with half-maximal inhibition for calcineurin at 0.16 units per mL. The inhibitory effect of calcineurin could be blocked by the pyrethroid deltamethrin, indicating inhibition of SV channels by calcineurin via dephosphorylation. A model is discussed in which vacuolar Ca2+ release through SV channels is subject to both positive feedforward and negative feedback control through cytosolic Ca2+ and dephosphorylation, respectively.     

Mathematical model of the neonatal mouse ventricular action potential

Therapies for heart disease are based largely on our understanding of the adult myocardium. The dramatic differences in action potential (AP) shape between neonatal and adult cardiac myocytes, however, indicate that a different set of molecular interactions in neonatal myocytes necessitates different treatment for newborns. Computational modeling is useful for synthesizing data to determine how interactions between components lead to systems-level behavior, but this technique has not been used extensively to study neonatal heart cell function. We created a mathematical model of the neonatal (day 1) mouse myocyte by modifying, on the basis of experimental data, the densities and/or formulations of ion transport mechanisms in an adult cell model. The new model reproduces the characteristic AP shape of neonatal cells, with a brief plateau phase and longer duration than the adult (action potential duration at 80% repolarization = 60.1 vs. 12.6 ms). The simulation results are consistent with experimental data, including 1) decreased density and altered inactivation of transient outward K+ currents, 2) increased delayed rectifier K+ currents, 3) Ca2+ entry through T-type as well as L-type Ca2+ channels, 4) increased Ca2+ influx through Na+/Ca2+ exchange, and 5) Ca2+ transients resulting from transmembrane Ca2+ entry rather than release from the sarcoplasmic reticulum (SR). Simulations performed with the model generated novel predictions, including increased SR Ca2+ leak and elevated intracellular Na+ concentration in neonatal compared with adult myocytes. This new model can therefore be used for testing hypotheses and obtaining a better quantitative understanding of differences between neonatal and adult physiology.     

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.     

Time dependence of the calcium-activated potassium current.

We investigated the dependence of the kinetics of the Ca2+-activated K+ current of the molluscan neuron soma upon membrane potential. The K+ current was activated by intracellular Ca2+ ion injection in neurons with blocked inward Na+ and Ca2+ currents. The difference between currents was measured with brief pulses (less than 100 ms) before and immediately after Ca2+ injection and was used as the Ca2+ activated K+ current at difference membrane potentials. The results in normal (10 mM) and in high (200 nM) external K+ show that the time-course of the Ca2+-activated K+ current depends upon membrane voltage and that the current activates more rapidly with membrane depolarization.     

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (approximately 55%) in mammalian and amphibian channels. Two amplitudes, differing by approximately 35%, were found in amphibian channel studies, probably corresponding to alpha and beta RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (approximately 40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.     

Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system.

In contracting cardiac myocytes, the rapid changes in cytosolic and nuclear Ca2+ make it difficult to determine whether the nuclear Ca2+ transient is caused by diffusion from the cytosol or by Ca2+ release channels on the inner nuclear membrane, or both. The propagation mechanism in the nucleoplasm also remains unknown. We have developed an ultra-fast Nipkow confocal imaging system able to acquire two-dimensional images at approximately 4 ms/full frame speed and employed it to analyze Ca2+ waves and the dynamics of the cytosolic and nuclear Ca2+ transients after electrical stimulation of cardiac myocytes. The pattern of nuclear Ca2+ upon stimulation was well described by a mathematical model of Ca2+ diffusion across the nuclear envelope. No evidence of Ca2+ release from perinuclear Ca2+ stores was obtained. The Ca2+ diffusion constant appeared to change during contraction, with essentially free diffusion of Ca2+ through nuclear pore complexes at low cytosolic Ca2+ and partially restricted diffusion at high cytosolic Ca2+. The Ca2+ in the nucleoplasm propagated by diffusion and no Ca2+ release phenomena were seen in the nucleus.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.