Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage- clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4- aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.     

Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra

Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could also be identified. The relevance of such conductance mechanisms in the generation of the light response in Lima photoreceptors is discussed.     

Action of exogenic acetylcholine on the potassium currents of frog motor nerve endings

Using nerve-muscle preparations of the cutaneous pectoris muscle of the frog, and recording extracellular evoked electrical responses from a nerve ending (NE), we have studied the mechanisms by which exogenic acetylcholine (ACh) influences the ion currents of a motor nerve ending. We have established that ACh in concentrations of 0.1–0.6 mmoles/liter causes an increase in the third phase of the NE response, and in concentrations of 0.7–2.0 mmoles/liter, suppresses it. We have found that tubocurarine and atropine do not block the effects of ACh. It has been shown that the ACh-induced increase in the amplitude of the third phase of the NE response is seen against a background of calcium channel blockers, and it can be eliminated by 4-aminopyridine (4-AP) and tetraethylammonium. The inhibitory effect of ACh on the third phase of the NE response is not present in calcium-free solutions, nor when the calcium current is blocked. A discussion is given of the mechanisms of exogenic and endogenic ACh action on the potential-dependent and calcium-activated potassium current of the NE.S. V. Kurashov Medical Institute, Ministry of Health of the Russian Federation, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 678–683, November–December, 1992.     

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3microM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3microM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10microM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30microM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30microM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.     

The control of chick myoblast fusion by ion channels operated by prostaglandins and acetylcholine

Chick myoblast fusion in culture was investigated using prostanoid synthesis inhibitors to delay spontaneous fusion. During this delay myoblast fusion could be induced by prostaglandin E1 (PGE1), by raising extracellular potassium and by addition of carbachol. Carbachol-induced fusion, but not PGE-induced fusion, was prevented by the acetylcholine receptor blocker alpha-bungarotoxin. Fusion induced by any of these agents was prevented by the Ca channel blockers lanthanum and D600. The threshold for potassium-induced fusion was 7-8 mM; maximal fusion occurred at 16-20 mM. Low extracellular potassium inhibited spontaneous fusion. Intracellular potassium in fusion competent myoblasts was 101 m-moles/l cell. Calcium flux measurements demonstrated that high potassium increased calcium permeability in fusion-competent myoblasts. A 30-s exposure to high potassium or PGE1 was sufficient to initiate myoblast fusion. Anion-exchange inhibitors (SITS and DIDS) delayed spontaneous myoblast fusion and blocked fusion induced by PGE1 but not carbachol. Blocking the acetylcholine receptor shifted the dose-response relation for PGE-induced fusion to higher concentrations. PGE1-induced fusion required chloride ions; carbachol-induced fusion required sodium ions. Provided calcium channels were available, potassium always induced fusion. We conclude that myoblasts possess at least three, independent pathways, each of which can initiate myoblast fusion and that the PGE-activated pathway and the acetylcholine receptor-activated pathway act synergistically. We suggest that fusion competent myoblasts have a high resting membrane potential and that fusion is controlled by depolarization initiated directly (potassium), by an increase in permeability to chloride ions (PGE), or by activation of the acetylcholine receptor (carbachol); depolarization triggers a rise in calcium permeability. The consequent increase in intracellular calcium initiates myoblast fusion.     

Ionic currents in avian granulosa cells

Transmembrane ionic currents have been recorded in single granulosa cells from the laying hen using the whole-cell patch-clamp technique. Under voltage-clamp conditions, depolarizing voltage steps evoked currents composed of a fast inactivating inward component and a delayed outward component. The former was activated at voltages more positive than -50 mV and was fully inactivated within 500 ms. It was blocked by D600 (methoxyverapamil) and by cobalt, suggesting that it is a calcium current. The latter displayed inward rectification and did not inactivate during long duration pulses. It was blocked by tetraethylammonium indicating that it is a potassium current. This is the first evidence of the existence of potassium and calcium transmembrane currents in granulosa cells.     

Properties of an acetylcholine-induced conductance in the rabbit atrial myocardium

Using a conventional microelectrode technique, action potentials (A.P.) recorded from the isolated left atrial trabeculae of the rabbit were analyzed. The membrane current during A.P. was reconstructed. In spite of an extracellular Ca2+-deficiency and the application of verapamil, acetylcholine (ACh) reduced the A.P. duration by inducing an outward current IACh. This current was blocked by atropine (10-6 M). A Nernst-plot of the reversal potential at different K+-concentrations showed a slope of 58.5 mV for a 10-fold change in concentration. After pacing pauses longer than 10 s an inward going (anomalous) rectification (A.R.) for IACh occurred. Increasing the duration of the pacing pauses the rectification was more accentuated. During a constant pacing the A.R. for IACh disappeared. ACh did not modify the A.R. The maximum slope conductance for IACh was dependent on the extracellular concentration of ACh (0.035 mS x cm-2 at 20 microM ACh, 0.012 mS x cm-2 at 0.2 microM ACh). The experimental results are discussed, using the model of an ACh-induced potassium channel. The channel should be related to the muscarinic receptor of the atrial myocardium.     

Calcium influx in contracting and paralyzed frog twitch muscle fibers

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.     

The action of Avermectin (MK 936) on identified central neurones from Helix and its interaction with acetylcholine and gamma-aminobutyric acid (GABA) responses

Intracellular recordings were made from identified neurones in the suboesophageal ganglionic mass of the snail, Helix aspersa. Avermectin, MK 936, 0.01-1.0 microM, induced an outward current in certain neurones. The size of this current varied from one cell type to another. This direct effect of Avermectin occurred irrespective of whether the neurones were sensitive to GABA or not and was generally irreversible. Avermectin, 0.1 microM, reduced the chloride mediated inhibitory GABA response and potentiated the largely sodium mediated excitatory GABA response. Avermectin, 0.1 microM, reduced the chloride mediated acetylcholine inhibitory response and potentiated the sodium mediated excitatory acetylcholine response. In neurones which showed a biphasic response to acetylcholine, Avermectin enhanced the excitatory and depressed the inhibitory component. It is concluded that Avermectin can interact with chloride ionophores to induce an outward current and can reduce chloride mediated responses associated with acetylcholine and GABA.     

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.     

Influence of platelet-activating factor on leukotriene D4-induced contractions of the guinea pig parenchymal strip

Platelet-activating factor (PAF) and sulphidopeptide leukotrienes, such as leukotriene D4 (LTD4), are potent constrictors that are probably released simultaneously in a variety of inflammatory respiratory events. The purpose of the present study was to determine whether LTD4-induced contractions of guinea pig parenchymal lung strips (GPPS) are modified in the presence of PAF. The contractile responses of isolated GPPS to cumulative doses of LTD4, acetylcholine, histamine, and potassium chloride in the presence of PAF (0.1 nM, 0.1 microM) were compared with parallel controls. There was no significant alteration of the response to acetylcholine and potassium chloride and the PAF-induced inhibition of the response to histamine, although significant, was not concentration dependent. In contrast, PAF in a concentration range from 0.1 nM to 1.0 microM caused a marked, concentration-dependent reduction of LTD4-induced contractions. Pretreatment with the PAF receptor antagonist, BN52021, prevented the attenuation of LTD4-induced contraction by PAF. The attenuation of LTD4-induced contraction by PAF was also prevented by pretreatment with indomethacin or with the thromboxane synthase inhibitor U63,557A, but not by pretreatment with the lipoxygenase inhibitors BW755c or nordihydroguaiaretic acid. Thus inhibition of LTD4-induced GPPS contraction by PAF is receptor dependent and probably secondary to thromboxane generation. The respiratory smooth muscle response to leukotrienes may be modified significantly by concomitant PAF release.     

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.     

Effects of calcium ionophore A23187 and calcium antagonists on 32Pi incorporation into polyphosphoinositides of rat cortical synaptosomes

The role of Ca2+ on 32Pi incorporation into polyphosphoinositides (PPI) of rat cortical synaptosomes was studied. Stimulation of muscarinic receptor by carbachol (1 mM) resulted in a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphophaphate (TPI) and phosphatidylinositol-4-phosphate (DPI), and an increase in 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA), whereas no significant effect on other membrane phospholipids was found. This response could be blocked by atropine (1 microM). The stimulatory effect of carbachol required Ca2+ in the medium; the presence of 0.5 mM EGTA blocked the effect of carbachol on PPI turnover completely. Calcium ionophore A23187, at 1 microM, had a similar effect on PPI turnover by carbachol (1 mM). At higher concentrations (10-100 microM) of A23187, the PPI turnover rate was much enhanced. Depolarization of the membrane by high potassium (60 mM) in the presence of calcium resulted in an enhanced PPI turnover, which was similar to the results of the carbachol (1 mM) effect but to a lesser extent. Calcium antagonists, diltiazem and trifluoperazine, at 10 microM could block the carbachol effect on 32Pi incorporation into PPI in this preparation. Our results suggest that the enhancement of PPI turnover in rat cortical synaptosomes by carbachol, calcium ionophore or high potassium requires Ca2+, and it can be blocked by compounds which interfere with the availability of this ion, such as EGTA or calcium antagonists.     

Characterization of nifedipine-resistant calcium current in neonatal rat ventricular cardiomyocytes

Calcium current was recorded from ventricular cardiomyocytes of rats at various stages of postnatal development using the whole cell patch-clamp technique. In cultured 3-day-old neonatal cells, the current carried by Ca(2+) or Ba(2+) (5 mM) was not completely inhibited by 2 microM nifedipine. A residual current was activated in the same voltage range as the L-type, nifedipine-sensitive Ca(2+) current, but its steady-state inactivation was negatively shifted by 16 mV. This nifedipine-resistant calcium current was not further inhibited by other organic calcium current antagonists such as PN200-110, verapamil, and diltiazem nor by nickel, omega-conotoxin, or tetrodotoxin. It was completely blocked by cadmium and increased by isoproterenol and forskolin. This current was >20% of total calcium current in ventricular myocytes freshly isolated from neonatal rats, and it decreased during postnatal maturation, disappearing at the adult stage. This suggests that this current could be caused by an isoform of the L-type calcium channel expressed in a way that reflects the developmental stage of the rat heart.     

Intestinal zinc uptake in freshwater rainbow trout: evidence for apical pathways associated with potassium efflux and modified by calcium

Understanding the mechanisms of intestinal zinc uptake in fish is of considerable interest from both nutritional and toxicological perspectives. In this study, properties of zinc transport across the apical membrane of freshwater rainbow trout intestinal epithelia were examined using right-side-out brush border membrane vesicles (BBMV's). Extravesicular calcium was found to have complex actions on zinc uptake. At a low zinc concentration of 1 microM, calcium (0.1-2 mM) significantly stimulated zinc uptake. In contrast, calcium inhibited zinc uptake at higher zinc levels (100 microM). Lanthanum and cadmium in the external medium did not block zinc uptake, suggesting that interactions between zinc and calcium were not exerted at a calcium channel. Copper also failed to exercise any inhibitory action. Zinc association with the BBMV's was enhanced by an outward potassium gradient. This stimulatory effect was only present at a zinc concentration of 100 microM. The potassium channel blocker, tetraethylammonium chloride inhibited zinc uptake at this relatively high zinc concentration, suggesting the presence of a low affinity zinc uptake pathway linked to potassium efflux. The present study provides evidence that the mechanism of intestinal zinc uptake in rainbow trout is pharmacologically very different from that of the piscine gill and the mammalian intestine.     

Functional Aspects of Calcium Channels of Splanchnic Neurons and Chromaffin Cells of the Rat Adrenal Medulla

Effects of the inorganic calcium channel blockers zinc, manganese, cadmium, and nickel on secretion of catecholamines from the perfused adrenal gland of the rat were investigated. Secretion of catecholamines evoked by splanchnic nerve stimulation (1 and 10 Hz) was not affected by nickel (100 microM), partially blocked (50%) by cadmium (100 microM), and almost completely blocked (90%) by zinc (1 mM) or manganese (2 mM). A combination of nickel and cadmium inhibited nerve stimulation-evoked secretion by 80-90%. Catecholamine secretion evoked by direct stimulation of chromaffin cells by acetylcholine (50 micrograms), nicotine (5 microM), muscarine (50 micrograms), and K+ (17.5 mM) was not blocked by either cadmium, nickel, or their combination. However, zinc and manganese almost abolished nicotine- and K(+)-evoked secretion of catecholamines. None of the above agents had any effect on the secretion evoked by muscarine. Acetylcholine-evoked secretion of catecholamines was only partially reduced (50%) by zinc and manganese. We draw the following conclusions from the above findings: (a) cadmium plus nickel selectively blocks the calcium channels of splanchnic neurons but has no effect on calcium channels of the chromaffin cells; (b) zinc and manganese do not discriminate between calcium channels of neurons and calcium channels of chromaffin cells; (c) partial inhibition of acetylcholine-evoked secretion by inorganic calcium channel blockers is consistent with the idea that activation of nicotinic receptors increases Ca2+ influx, and activation of muscarinic receptors mobilizes intracellularly bound Ca2+, which is not affected by calcium channel blockers.     

Cobalt activates potassium conductance in the plasma membrane of cultured renal epithelioid (MDCK)-cells

Cobalt has been shown to stimulate sodium transport across the distal nephron of the newt kidney. The mechanism of this action remained elusive. The present study has been performed to test for effects of cobalt on electrical properties of cultured subconfluent kidney (MDCK)-cells: cobalt (10 microM) leads to a rapid, sustained and reversible hyperpolarization of the cell membrane, paralleled by an increase of the potassium selectivity and a decrease of the resistance. Thus, cobalt increases the potassium conductance of the cell membrane. The half-maximal effect is elicited by approx. 1 microM. At extracellular calcium concentration reduced to less than 0.1 microM, cobalt (10 microM) leads to a transient hyperpolarization, which can be elicited only once. Thus, cobalt enhances the potassium conductance in a calcium dependent way. At higher concentrations (100 microM) cobalt hyperpolarizes the cell membrane only transiently even in the presence of extracellular calcium. Furthermore 100 microM cobalt interferes with ATP-induced hyperpolarization, which is known to result from calcium mediated activation of K+ channels. Thus, 100 microM cobalt may inhibit ATP-stimulated calcium entry into the cell.     

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.