Depolarization shifts the voltage dependence of cardiac sodium channel and calcium channel gating charge movements

In cardiac ventricular myocytes, membrane depolarization leads to the inactivation of the Na channel and Ca channel ionic currents. The inactivation of the ionic currents has been associated with a reduction of the gating charge movement (“immobilization”) which governs the activation of Na channels and Ca channels. The nature of the apparent “immobilization” of the charge movement following depolarization was explored in embryonic chick ventricular myocytes using voltage protocols applied from depolarized holding potentials. It was found that although all of the charge was mobile following inactivation, the voltage dependence of its movement was shifted to more negative potentials. In addition, the shift in the distribution of the Na channel charge could be differentiated from that of the Ca channel charge on the basis of kinetic as well as steady-state criteria. These results suggest that the voltage-dependent activation of Na channel and Ca channel charge movements leads to conformational changes and charge rearrangements that differentially bias the movements of these voltage sensors, and concomitantly produce channel inactivation. Received: 26 September 1995 / Accepted: 7 November 1995     

Intramembrane charge movement in developing skeletal muscle cells from fetal mice

The development of intramembrane charge movement was studied in freshly isolated skeletal muscle cells from 13- to 19-day-old mouse fetuses. Charge movement was present in myotubes from 13-day-old fetuses. The relationship between charge movement and membrane potential could be described by a two-state Boltzmann equation. The amount of maximum charge movement (Q _max) increased substantially with the age of the fetuses from 2.84±0.39 nC/F (n=10) at day 13 to 10.01±0.97 nC/F (n=15) at day 19. Nifedipine (1 M) consistently reduced Q _max by 33±2% (n=37) of the control value at each age studied. Increasing the concentration of nifedipine to 20 M had no further effect, suggesting that the charge movement in developing myotubes consists of at least two components: a nifedipine-sensitive charge movement (Q _ns) and a nifedipine-resistant one (Q _nr). Both Q _ns and Q _nr increased exponentially with a distinct enhancement of rate at day 16.     

The effects of calcium and calcium channel blockers on sodium pump

The effects of 10 mM Ca²⁺ and Ca²⁺ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na⁺, K⁺ pump activity of mouse diaphragm muscle fibres enriched with Na⁺ were compared with the changes in cytosolic [Ca²⁺]. The electrogenic Na⁺ pump activity produced by adding K⁺ to muscles previously bathed for 4 h in a K⁺-free, 2-mM [Ca²⁺] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca²⁺. Verapamil 10^–5M, 10^–5M diltiazem and 10^–7 M flunarizine effectively prevented the effect of elevated [Ca²⁺]. At these concentrations, these drugs did not affect the K⁺-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca²⁺] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]2-(2-amino 5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261±6 nM. After 4 h in a Liley K⁺-free, 2 mM [Ca²⁺] solution, the cytosolic [Ca²⁺] increased to 314±28 nM. Increase in [Ca²⁺] from 2 to 10 mM caused a twofold increase of cytosolic [Ca²⁺] to 637±26 nM. This rise was, like the Ca²⁺-induced inhibition of electrogenic pump, prevented by 10^–5 M verapamil, 10^–5M diltiazem and 10^–7 M flunarizine. The results suggest that substances which block Ca²⁺ entry into the cell prevent the Ca²⁺ induced inhibition of the Na⁺ pump.     

Voltage-dependent properties of macroscopic and elementary calcium channel currents in guinea pig ventricular myocytes

Whole-cell Ca channel currents were recorded from guinea pig ventricular myocytes that were internally perfused with Cs solution and bathed in solutions containing 3.6 mM Ca, 3.6 mM Ba or 90 mM Ba (34° C). Single Ca channel currents were recorded from cell-attached membrane patches of similar myocytes; the patch pipettes contained a 90 mM Ba solution. 1. Although the shape of the whole-cellI–V relation was independent of the bathing solution, this was not the case with the location of the inward current maximum (V _peak);V _peak in 90 mM Ba was about 30 mV positive toV _peak in 3.6 mM Ba. 2. The activation and inactivation of whole-cell currents were voltage dependent. Compared to the voltage dependencies in 3.6 mM Ba, those in 90 mM Ba were shifted by about 30 mV to the right, suggesting a neutralization of surface charges. 3. Observations compatible with the ion permeation model proposed by Hess and Tsien (1984) included (a) a depression of current during Ca/Ba solution exchange, (b) a high divalent to monovalent ion permeability, and (c) rectification of the outward limb of theI–V relation. 4. Estimated current densities atV _peak were similar for myocytes in 3.6 mM Ca and 3.6 mM Ba, and about 10 times larger in 90 mM Ba. 5. Average currents (I ^*) calculated from ensembles of records of single Ca channel current had voltage-dependent time courses resembling those of whole-cellI _Ba (90 mM). 6. Single-channelI ^*–V relations were superimposable on whole-cellI–V curves suggesting that voltage-dependent single-channel parameters (probability of opening, elementary current amplitude) can be related to the voltage-dependent macroscopic current parameters (activation, instantaneousI–V relation) when scaled by channel number. 7. The density of Ca channels in myocytes was calculated from whole-cellI _Ba (90 mM) and average current through single channels. The outcome, 3–5 channels/m², agrees with two other recent estimates (Tsien et al. 1983; Lux and Brown 1984). However, it is difficult to reconcile with the much lower density that one would forecast from the frequency of functional channel observation in myocyte membrane patches (Pelzer et al. 1985c).Supported by the Deutsche Forschungsgemeinschaft (DFG), SFB 38 (Membranforschung), project G1, and the Medical Research Council (Canada)     

Voltage- and concentration-dependent effects of lidocaine on cardiac Na channel and Ca channel gating charge movements

The effects of lidocaine, a local anesthetic and cardiac antiarrhythmic agent, were studied on cardiac nonlinear Na channel and Ca channel charge movements (gating currents) of 17-day-old embryonic chick ventricular myocytes. Gating currents were recorded following the blockade of all ionic currents and the subtraction of the linear capacity currents (-P/5). From a holding potential of –100 mV the ON charge movement (Q _ON) displayed two kinetic components: a rapidly decaying component associated with Na channel gating, and a slower component associated with Ca channel gating. A depolarizing prepulse to –50 mV for 125 ms reduced the fast component of Q _ON, with little effect on the slower component. Similarly, 20 M lidocaine also reduced the fast component of Q _ON (Na channel charge movement) and had little effect on the slower component (Ca channel charge movement). Higher concentrations of lidocaine (125 M) reduced both the fast and the slower components of Q _ON. The effects of either a prepulse to –50 mV, or 20 M lidocaine on the steady-state Q _ON/V _m relationship were nearly identical. These results suggest that lidocaine blocks cardiac Na (ionic) currents by a reduction in the availability of Na channel charge movement (Q _ON), and that this reduction is similar to that produced by voltage-dependent inactivation.     

Presynaptic regulation of hippocampal acetylcholine release is unaffected by calcium channel blockers and intracellular calcium chelation

Endogenously produced nitric oxide (NO) was monitored in exhaled air from ovalbumin-sensitized and pentobarbital anaesthetized guinea-pigs. Stable levels of nitric oxide were detected in exhaled air over a 30-min control period in each experiment (9.2 ±1.4 parts per billion, [ppb]). Insufflation pressure and NO in exhaled air immediately increased, in a dose dependent manner, in response to challenge with nebulized allergen (Ovalbumin, 0.1–10 mg). Indomethacin (5 mg kg^-1) augmented the allergen-induced increases in insufflation pressure and NO. Fifteen min after the challenge the insufflation pressure remained elevated while NO in exhaled air had dropped below control levels. The increase in insufflation pressure induced by inhalation of PGF_2a(5 μg) was accompanied by an increase in nitric oxide in exhaled air, which however was significantly less than the increase in NO induced by allergen challenge. The results suggest a role for NO mechanisms in asthma.     

Effect of L-type calcium channel antagonists on spermine-induced CNS excitation in vivo

The ability of nitrendipine, nisoldipine, verapamil and gabapentin to inhibit the development of CNS excitation induced by spermine was assessed in mice. Injection of an excitotoxic dose of spermine (100 μg, i.c.v.) in mice results in worsening tremor that culminates in the development of a fatal tonic convulsion within 8 h of spermine administration. The dihydropyridines, nitrendipine and nisoldipine, which are L-type calcium channel antagonists acting at the 1 subunit, inhibited the development of spermine-induced effects. Verapamil, which also acts at the 1 subunit of the L-type calcium channel, also inhibited the development of spermine-induced CNS excitation. Gabapentin, a postulated L-type calcium channel antagonist interacting at the 2δ subunit, did not inhibit the development of spermine-induced effects. These results show that antagonists of the 1 subunit of L-type calcium channels can effectively inhibit the effects of spermine in vivo. This may highlight the importance of L-type calcium channels in spermine action.     

The rate of tetanic relaxation is correlated with the density of calcium ATPase in the terminal cisternae of thyrotoxic skeletal muscle

We have studied voltage-dependent calcium channels in the A7r5 smooth muscle cell line by measuring the high-affinity binding of radiolabelled dihydropyridines (DHPs), whole-cell and single-channel currents in patchclamped cells, as well as cytosolic calcium ([Ca²⁺]_i) in fura-2-loaded cell suspensions and monolayers. Intact A7r5 cells express saturable, high-affinity, voltage-sensitive DHP binding sites with pharmacological properties characteristic of L-type calcium channels. When cells were voltage clamped in the whole-cell configuration with near normal intra- and extracellular solutions, a DHP-sensitive inward current resembling the L-type calcium current was dominant. With barium (10 mM) as the charge carrier, peak inward currents were typically recorded at test potentials between 0 and +20 mV. Currents were blocked by extracellular cadmium with a half-maximal inhibitory concentration of 1 M. Isoproterenol (1 M.) or forskolin (10 M) increased currents in approximately half of the cells tested. Forskolin (10 M) increased single-channel activity in five of eight cell-attached patches. After cells had been quiescent for several weeks, cell suspensions showed changes in resting [Ca²⁺]_i in response to DHPs and increased potassium. Most confluent monolayers of cells showed spontaneous transient elevations in [Ca²⁺]_i. Bath application of Bay K 8644 increased the frequency and magnitude of these [Ca²⁺]_i transients, whereas nifedipine abolished the transients. These data suggest that the [Ca²⁺]_i transients were due to synchronous action potentials in electrically coupled cell monolayers.     

Cilnidipine, an L/N-type calcium channel blocker prevents acquisition and expression of ethanol-induced locomotor sensitization in mice

Several evidences indicated the involvement of L- and N-type calcium channels in behavioral effects of drugs of abuse, including ethanol. Calcium channels are implicated in ethanol-induced behaviors and neurochemical responses. Calcium channel antagonists block the psychostimulants induced behavioral sensitization. Recently, it is demonstrated that L-, N- and T-type calcium channel blockers attenuate the acute locomotor stimulant effects of ethanol. However, no evidence indicated the role of calcium channels in ethanol-induced psychomotor sensitization. Therefore, present study evaluated the influence of cilnidipine, an L/N-type calcium channel blocker on acquisition and expression of ethanol-induced locomotor sensitization. The results revealed that cilnidipine (0.1 and 1.0μg/mouse, i.c.v.) attenuates the expression of sensitization to locomotor stimulant effect of ethanol (2.0g/kg, i.p.), whereas pre- treatment of cilnidipine (0.1 and 1.0μg/mouse, i.c.v.) during development of sensitization blocks acquisition and attenuates expression of sensitization to locomotor stimulant effect of ethanol. Cilnidipine per se did not influence locomotor activity in tested doses. Further, cilnidipine had no influence on effect of ethanol on rotarod performance. These results support the hypothesis that neuroadaptive changes in calcium channels participate in the acquisition and the expression of ethanol-induced locomotor sensitization.     

Differentiation-dependent alterations in the extracellular ATP-evoked calcium fluxes of cultured skeletal muscle cells from mice

Although extracellular adenosine triphosphate (ATP) has been generally accepted as the regulator of cellular differentiation, the relative contribution of the various purinoreceptor subtypes to purinergic signalling at distinct stages of skeletal muscle differentiation is still poorly understood. Here we measured extracellular ATP-evoked changes in intracellular calcium concentration and surface membrane ionic currents (I _ATP), calculated the calcium flux (FL) entering the myoplasmic space and compared these parameters at different stages of differentiation on cultured mouse myotubes. The ATP-evoked FL displayed an early peak and then declined to a steady level. With differentiation, the early peak became separated from the maintained component and was absent on mature myotubes. Repeated ATP applications caused desensitization of the response in both immature and differentiated myotubes, owing mainly to the reduction of the early peak of FL in the former and to a decline of both components in the latter group of cells. Depolarization of the cell or removal of external calcium suppressed the early peak. I _ATP showed no inactivation, and its voltage dependence displayed strong inward rectification. The concentration dependence of I _ATP can be fitted using a Hill equation, yielding an EC₅₀ of 56 μM. Results are consistent with the parallel activation of both P2X and P2Y receptors.     

钙通道阻滞剂对缺氧/复氧心肌细胞的保护作用

目的:观察钙通道阻滞剂对缺氧/复氧(A/R)心肌细胞的保护作用。方法:原代培养大鼠心肌细胞分为A/R、A/R+硝苯地平(Nif)、A/R+钌红(Ru)+肝素(Hep)和对照4组。检测各组心肌细胞内钙浓度(i)、细胞活力、ATP含量及孵育液中乳酸脱氢酶(LDH)含量、[³H]-亮氨酸([3H]³H-Leu)掺入量、丝裂素活化蛋白激酶(MAPK)和蛋白激酶C(PKC)活性。结果:A/R+Nif和A/R+Ru+Hep组心肌细胞i、孵育液中LDH均显著低于A/R组(P<0.01);细胞活力、ATP含量、PKC和MAPK活性、³H-Leu掺入量显著高于A/R组(P<0.05或P<0.01)。结论:阻断心肌细胞外Ca²⁺内流及内贮Ca²⁺的释放,可通过减轻A/R介导的细胞Ca²⁺超载而对心肌细胞起保护作用。     

Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker,verapamil

Summary We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculline-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 M) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50–150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.     

胰岛素作用的跨膜钙离子流变化与人肝癌细胞增殖的关系

目的:揭示胰岛素影响人肝癌细胞增殖的机制。方法:利用人肝癌细胞系(HepG-2)为靶细胞,以原子吸收光谱技术检测胰岛素对靶细胞跨膜钙离子流影响,同时结合钙离子拮抗剂(isoptin)的作用来研究靶细胞的增殖变化。结果:胰岛素能明显促使靶细胞跨膜钙离子内流而改变其恒态,并对靶细胞增殖有明显的促进作用;而钙离子拮抗剂却明显抑制其细胞的增殖。结论:胰岛素促使人肝癌细胞增殖的机制与其细胞跨膜钙离子流恒态的变化密切相关。     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

The slow inward calcium current is responsible for a part of the contraction of patch-clamped rat myoballs

The slow inward calcium current and the contractile response were simultaneously recorded in voltage clamped (whole cell patch clamp recording) rat myoballs in primary culture.The shape of the contraction(T)/potential(V) relationship and the application of the inorganic calcium channel blocker cadmium (1.5 mM), which suppresses a part of the contractile activity, demonstrate the existence of two components of contraction. One of them is related to the slow calcium current.     

Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion

Zn²⁺ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca²⁺ independent; in fact, in the absence of extracellular Ca²⁺, the secretory effects of Zn²⁺ were enhanced. At low concentrations (3–10 M), Zn²⁺ enhanced the secretory responses to 10-s pulses of 100 M 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K⁺. In the presence of DMPP, secretion was increased 47% above controls and in high-K⁺ solutions, secretion increased 54% above control. These low concentrations of Zn²⁺ did not facilitate the whole-cell Ca²⁺ (I _Ca) or Ba²⁺ (I _Ba) currents in patch-clamped chromaffin cells. Higher Zn²⁺ concentrations inhibited the currents (IC₅₀ values, 346 M for I _Ca and 91 M for I _Ba) and blocked DMPP- and K⁺-evoked secretion (IC₅₀ values, 141 and 250 M, respectively). Zn²⁺ permeated the Ca²⁺ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba²⁺, Ca²⁺, Sr²⁺ and Zn²⁺ were 991, 734, 330 and 7.4 pA, respectively. Zn²⁺ entry was also evidenced using the fluorescent Ca²⁺ probe fura-2. This was possible because Zn²⁺ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca²⁺, i.e. 360 nm. There was a slow resting entry of Zn²⁺ which was accelerated by stimulation with DMPP or high-K⁺ solution. The entry of Zn²⁺ was concentration dependent, slightly antagonized by 1 mM Ca²⁺ and completely blocked by 5 mM Ni²⁺. The entry of Ca²⁺ evoked by depolarization with high-K⁺ solution was antagonized by Zn²⁺. We conclude that inhibition by Zn²⁺ of evoked catecholamine secretion is associated with blockade of Ca²⁺ entry through Ca²⁺ channels recruited by DMPP or K⁺. However, the facilitation of secretion observed at low Zn²⁺ concentrations, or in the absence of Ca²⁺, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn²⁺ permeates the bovine chromaffin cell Ca²⁺ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn²⁺ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn²⁺ to fura-2 and the modification of its fluorescence spectrum.     

Localization of L-type calcium channel CaV1.3 in cat lumbar spinal cord – with emphasis on motoneurons

Voltage-dependent persistent inward currents (PICs) which underlie the plateau potentials are an important intrinsic property of spinal motoneurons. Electrophysiological experiments have indicated that a subtype of the low threshold L-type calcium channel, Ca_V1.3, mediates this current. In mouse and turtle lumbar spinal cord it has been shown that these channel proteins are mainly found on motoneuron dendrites. In the present study we have used immunohistochemistry to locate these channels in lumbar spinal neurons, especially motoneurons, of the cat. The results indicate that Ca_V1.3 immunoreactivity was unevenly distributed among the laminae of the spinal grey matter. The small neurons in superficial dorsal horn (laminae I–III) were sparsely and weakly labelled, while large neurons in ventral horn were frequently and densely labelled. Groups of motoneurons in lamina IX that were immunoreactive to choline acetyltransferase also co-expressed Ca_V1.3. The immmunoreactivity was mainly associated with neuronal somata and proximal dendrites. Double staining with antibodies against Ca_V1.3 and MAP2 (a dendritic marker) showed that some fine fibres, which may include distal dendrites, were also labelled. These results in the cat spinal cord show some differences from studies in mouse and turtle motoneurons where the immunoreactivity against this channel was mainly localized to the dendrites.     

Nucleoplasmic reticulum is not essential in nuclear calcium signalling mediated by cyclic ADPribose in primary neurons

Nuclear calcium regulation is essential for controlling nuclear processes such as gene expression. Recent studies, mostly performed on immortalized or transformed cell lines, reported the presence of a nucleoplasmic reticulum (NR). It has been suggested that NR acts as a storage organelle having an important role in nuclear Ca²⁺ signalling. However, whether NR is present and necessary in primary neurons for generation of nuclear Ca²⁺ signalling has never been investigated. Here, we show, by confocal microscopy and by electronic microscopy, that nuclei in intact neurons or isolated nuclei are not endowed with NR. Finally, our experiments performed on isolated nuclei from Aplysia giant neurons show that the nuclear envelope acts as a functional Ca²⁺ store which can be mobilized by the second messenger cyclic ADPribose to elicit a nucleoplasmic Ca²⁺ elevation. Our study provides evidence that nuclear Ca²⁺ signals can be independent of the presence of NR in neurons.     

Relating ionisation of calcium chloride in saliva to bitterness perception

Saliva plays a role in the perception of bitter, sour and salty tastes that are presumed to be derived from the concentration of free cations or anions ions dissolved in saliva. The role of ionisation of calcium in bitter taste was studied by determining binding in vitro mixture of saliva and protein solutions and in spit. In vitro, the addition of whey to calcium chloride solutions increased the calcium binding, pH and viscosity. The addition of saliva to these mixtures, the increased calcium binding and the induced small changes in viscosity and pH were thought not to contribute significantly to bitterness perception. Nonstimulated saliva, at pH 7.5, contained about 5 mM calcium, of which about one third was ionised. The bitter threshold of fully ionised calcium chloride in water varied between 1 and 15 mM among individuals. In spit, after tasting whey, ionised calcium was found to have increased at low, but decreased at high, calcium concentrations and varied 30% among individuals. Bitterness was related, on average, to the concentration of ionised calcium and not to the total concentration of calcium in spit. A general explicative model based on the composition of bulk saliva is discussed in relation to perception threshold and the likely importance of saliva from von Ebner's gland.