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

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

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Perspective: The List of Potential Volume-sensitive Chloride Currents Continues to Swell (and Shrink)

The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca²⁺ approximately four times more efficiently than that of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs⁺ efflux and Ca²⁺ influx in recombinant NMDAR and Ca²⁺-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca²⁺ using whole-cell currents and fluorometric techniques (dye overload) with Cs⁺ internally and Ca²⁺ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca²⁺ and Cs⁺ as the reference ions, Ca²⁺ permeability (P_Ca/P_Cs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; P_Ca/P_Cs ≈ 6.1) and reduced at both higher (110 mM; P_Ca/P_Cs ≈ 2.6) and lower (0.18 mM; P_Ca/P_Cs ≈ 2.2) concentrations. P_Ca/P_Cs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca²⁺. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca²⁺ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca²⁺ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca²⁺ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca²⁺, whereas that of AMPAR channels only a single site.     

Ca<Superscript>2+</Superscript> current of frog vestibular hair cells is modulated by intracellular ATP but not by long-lasting depolarisation

Some aspects of Ca²⁺ channel modulation in hair cells isolated from semicircular canals of the frog (Rana esculenta) have been investigated using the whole-cell technique and intra and extracellular solutions designed to modify the basic properties of the Ca²⁺ macrocurrent. With 1 mM ATP in the pipette solution, about 60% of the recorded cells displayed a Ca²⁺ current constituted by a mix of an L and a drug-resistant (R2) component; the remaining 40% exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca-dependent manner. If the pipette ATP was raised to 10 mM, cells exhibiting the R1 current fraction displayed an increase of both the R1 and L components by ∼280 and ∼70%, respectively, while cells initially lacking R1 showed a similar increase in the L component with R1 becoming apparent and raising up to a mean amplitude of ∼44 pA. In both cell types the R2 current fraction was negligibly affect by ATP. The current run-up was unaffected by cyclic nucleotides, and was not triggered by 10 mM ATPγS, ADP, AMP or GTP. Long-lasting depolarisations (>5 s) produced a progressive, reversible decay in the inward current despite the presence of intracellular ATP. Ca²⁺ channel blockade by Cd²⁺ unmasked a slowly activating outward Cs⁺ current flowing through a non-Ca²⁺ channel type, which became progressively unblocked by prolonged depolarisation even though Cs⁺ and TEA⁺ were present on both sides of the channel. The outward current waveform could be erroneously ascribed to a Ca- and/or voltage dependence of the Ca²⁺ macrocurrent. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Characteristics of Ca2+-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin

We have studied the activities of Ca²⁺-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca²⁺-stimulated ATPase in sarcolemma exhibited two activation sites — one with low affinity (Km = 0.70 ± 0.2 mM; Vmax = 10.0 ± 2.2 mol Pi/mg/h) and the other with high affinity (Km = 0.16 ± 0.7 mM; Vmax = 4.6 ± 0.8 mol Pi/mg/h) for Mg²⁺ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca²⁺-stimulated ATPase without affecting its sensitivity to Ca²⁺ or Mg²⁺ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5–10 mM concentrations caused a 4–5 fold activation of Ca²⁺-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg²⁺ATP. DTT increased the affinity of the enzyme to Mg²⁺ATP at the high affinity site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca²⁺-stimulated ATPase to Ca²⁺. DTT protected the Ca²⁺-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca²⁺-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca²⁺-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca²⁺-stimulated ATPase activity in sarcolemmal preparations.     

A Ca2+-induced Ca2+ Release Mechanism Involved in Asynchronous Exocytosis at Frog Motor Nerve Terminals

The extent to which Ca²⁺-induced Ca²⁺ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca²⁺ ([Ca²⁺]_i) in presynaptic terminals (Ca²⁺-hump) in frog skeletal muscles over a period of minutes in a low Ca²⁺, high Mg²⁺ solution. Mn²⁺ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca²⁺ channels. MEPP-hump depended on extracellular Ca²⁺ (0.05–0.2 mM) and stimulation frequency. Both the Ca²⁺- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN⁻. Thus, Ca²⁺-hump is generated by the activation of CICR via ryanodine receptors by Ca²⁺ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca²⁺ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca²⁺-hump affected little MEPP frequency or [Ca²⁺]_i, but caused a quick increase (faster than MEPP- or Ca²⁺-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca²⁺ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca²⁺ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca²⁺ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.     

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.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.     

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

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

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca²⁺ release (“Ca²⁺ sparks”) through ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum and K_Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K_Ca channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca²⁺ spark. The amplitudes of the Ca²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K_Ca channel open probability (>10⁴-fold) during a Ca²⁺ spark is consistent with local Ca²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F_o) measured during a Ca²⁺ spark (mean 2.04 F/F_o or ∼310 nM Ca²⁺) appears to significantly underestimate the local Ca²⁺ that activates K_Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca²⁺ sparks are functionally coupled to K_Ca channels in the surface membrane, providing direct support for the idea that Ca²⁺ sparks cause STOCs.     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Rational method in the repetitive calcium oscillation measurement in wild type human epithelial kidney cells

Cells stimulated with physiological stimuli usually exhibit oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), a signal playing central roles in regulation of various cellular processes. For explicating their unknown mechanisms, studies are commonly conducted in single cells from several cell lines, in particular the human epithelial kidney (HEK293) cell line. However, [Ca²⁺]_i oscillating responses to agonists in vitro are found difficult to be induced and varied with different types of cells and agonists. This study shows that treatment of the wild type HEK293 cells with low concentrations of carbachol (1–10 μM), an agonist of the muscarinic receptor, resulted in non-oscillated but sustained [Ca²⁺]_i increase by loading the cells with 1 μM fura2/AM. However, repetitive and long lasting [Ca²⁺]_i oscillations could be induced in 31.1% of the tested cells loaded with 0.1 μM fura2/AM. Additionally, the occurrence of the typical Ca²⁺ spikes further increased to 47.2% and 60.7% when the Ca²⁺ concentration in the bathing medium was decreased from 1.8 mM to 1.5 mM and the medium temperature was set to 35 ± 1°C from 22 ± 2°C. Therefore, this study provides a useful approach for measuring [Ca²⁺]_i oscillatory response to relevant physiological stimulation in a wild type cell line through the adjustments of the concentrations adopted for the Ca²⁺ indicator and extracellular medium Ca²⁺ and of the temperature set for the experiment.