The purified protein that binds the K+ channel ligands dendrotoxin I and mast cell degranulating peptide can be phosphorylated by cAMP-dependent protein kinase and by an endogenous protein kinase, which may be a specific K+ channel kinase. Phosphorylations take place on the toxin-binding subunit, a polypeptide of 76-80 kDa. Phosphorylation by both kinases leads to activation of the reconstituted dendrotoxin-sensitive K+ channel.

Animals↗

PubMed1989

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.

Animals↗

PubMed1989

Calcium currents in single SA nodal cells of the rabbit heart studied with action potential clamp.

With a new method called "Action Potential-Clamp" (APC) we studied in single SA nodal cells the contribution of both transient and long lasting calcium currents (L-type and T-type) to the action potential. Action potentials were recorded by a computer and a representative cycle was subsequently repetitively replayed to the same cell under voltage clamp. Blockade of the L-type calcium current (D600) or T-type calcium current (nickel) revealed the quantitative and time related contributions of these currents to the action potential, since the blocked current is compensated by the clamp amplifier.

Action Potentials↗

PubMed1989

Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Dual action (stimulation, inhibition) of D600 on contractility and calcium channels in guinea-pig and cat heart cells.

1. We examined the effects of D600 (0.2-40 microM, generally 2 microM) on the following (i) developed tension in guinea-pig papillary muscles, (ii) calcium current (Ica) and tension in cat ventricular muscle strands, (iii) Ica in guinea-pig and cat ventricular myocytes, (iv) single Ca2+ channel currents carried by Ba2+ in cell-attached membrane patches of guinea-pig ventricular myocytes, and (v) Ba2+ currents through dihydropyridine (DHP)-binding sites (skeletal muscle) reconstituted into single functional Ca2+ channels in lipid bilayers. 2. In 27 of 140 preparations studied, D600 elicited a transient stimulation that preceded marked inhibition. The stimulation was normally of short duration (less than 5 min) and moderate strength (less than 50% increase). 3. D600 had no effect on the unit conductance of single cardiac Ca2+ channels. Stimulation was characterized by a decrease in the number of records with no openings (blanks) and an increase in the open-state probability of non-blanks (longer open times, shorter closed times). Inhibition began with an increase in the number of blanks and later included a curtailment of open times and a prolongation of closed times. The net effect after 9 min D600 was a 75% reduction in average current amplitude. 4. A similar pattern of changes in channel open and closed times produced enhancement and then depression of time-averaged open-state probability in single reconstituted channels. 5. Single Ca2+ channel current that was stimulated by adrenaline was only slightly depressed after 2 microM-D600 for 30 min. It may be that channel phosphorylation or Gs-protein activation following beta-receptor stimulation reduces channel affinity for D600. 6. Short-lived binding of D600 to a single inhibitory site may enhance association/activation of Gs-protein and thereby cause transient up-regulation prior to increased drug occupancy and inhibition. Alternatively, there may be separate stimulatory and inhibitory sites. One aspect of inhibition, the increased frequency of blanks, is attributed to a stabilization of the inactivated state; the other aspect, changes in fast kinetics, seems to require a different explanation.

Animals↗

PubMed1988

Angiotensin II-induced stimulation of voltage-dependent Ca2+ currents in an adrenal cortical cell line.

Biochemical studies suggest that stimulation of aldosterone secretion by angiotensin II involves activation of voltage-dependent Ca2+ channels. We used an adrenocortical cell line (Y1) to study the effect of angiotensin II on transmembranous currents. The hormone (1 nM to 1 microM) caused depolarization of the plasma membrane (from -35 to 10 mV) and elicited repetitive action potentials. Using the whole-cell clamp technique, we identified two types of voltage-dependent Ca2+ currents which differed with respect to their threshold potential and time course of inactivation. Angiotensin II (1 nM to 1 microM) stimulated a slowly inactivating Ca2+ current on average up to 1.7-fold whereas a fast inactivating Ca2+ current remained almost unaffected by the hormone. Ca2+ currents were not influenced by forskolin (1 microM) or intracellularly applied cAMP (50 microM). Pretreatment of cells with pertussis toxin abolished the hormonal stimulation of the slowly inactivating Ca2+ current but was without effect on control currents. The toxin ADP-ribosylated a single membranous peptide of 40 kd Mr. An antiserum raised against a synthetic peptide corresponding to a region common to all sequenced alpha-subunits of guanine nucleotide-binding proteins (G-proteins) and an antiserum raised against a peptide corresponding to a region of alpha-subunits of Gi-like G-proteins reacted with membranous 40 kd peptides, whereas an antiserum raised against a synthetic peptide corresponding to a region specific for the alpha-subunit of the G-protein, G0, failed to recognize a peptide in the 39 to 40 kd region.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1988

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.

Animals↗

PubMed1988

Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes.

The effects of a protein phosphatase inhibitor, okadaic acid (OA), were studied on membrane currents of isolated myocytes from guinea-pig cardiac ventricle. The whole-cell Ca2+ current (ICa) was recorded as peak inward current in response to test pulse to 0 mV. Extracellular application of OA (5-100 microM) produced an increase of ICa. The effect was markedly enhanced when the myocyte was pretreated with threshold concentrations of isoprenaline. ICa was increased from 11.3 +/- 0.8 microA cm-2 to 19.0 +/- 1.1 microA cm-2 (n = 4) by 5 microM-OA in the presence of 1 nM-isoprenaline. The delayed rectifier current was also slightly increased. Furthermore, the wash-out time of the beta-adrenergic increase of ICa was markedly prolonged by OA. The beta-adrenergic stimulation of cardiac Ca2+ current is thought to be mediated by cAMP-dependent phosphorylation. The present results strongly suggest that the effect of OA on ICa is related to inhibition of endogenous protein phosphatase activity which is responsible for the dephosphorylation process. By the isotope method, the inhibitory effect of OA on different types of phosphatase was compared. OA had a relatively high specificity to type 1-, and type 2A-phosphatases.

Animals↗

PubMed1988

A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes.

The decrease ('run-down') of the L-type Ca2+-current during long-lasting recordings with the whole-cell patch-clamp technique was examined in guinea pig ventricular myocytes. We have tested whether proteolysis is involved in the decay of the Ca2+-current by intracellular application of several concentrations of the Ca2+-dependent proteases calpain I and II, as well as their endogenous inhibitor calpastatin. The major finding was that calpain I and calpain II accelerated the Ca2+-current run-down in a concentration dependent manner, whereas calpastatin retarded it. These observations indicate that a proteolytic degradation of Ca2+-channels might be the reason for the run-down phenomenon.

Animals↗

PubMed1988

Intracellular N-methyl-D-glucamine modifies the kinetics and voltage-dependence of the calcium current in guinea pig ventricular heart cells.

The effects of internal substitution of the impermeant cation N-methyl-D-glucamine (NMG) for Cs ion on the properties of the Ca-current (L-type channel) were examined in single guinea pig cardiac myocytes with the whole-cell clamp technique. The properties of the cobalt-sensitive Ca current recorded in the presence of internal NMG or Cs were compared and the results were as follows. (1) The overall duration of the Ca-dependent slow action potential was markedly increased in the presence of internal NMG (6-fold at 0 mV) when compared to action potentials recorded with internal Cs. (2) The cobalt-sensitive Ca currents recorded with internal NMG or Cs had similar reversal potentials. However, in the presence of internal NMG, the maximum current density of the cobalt-sensitive Ca current was decreased and both the threshold and potential at which maximum current occurred were negatively shifted. (3) Voltage-dependence of steady-state activation, but not inactivation, of the cobalt-sensitive Ca current was shifted by -11.8 mV with internal NMG. (4) NMG increased the half-time of activation and inactivation of the cobalt-sensitive Ca current. The voltage-dependence of the half-time of inactivation was shifted by about -30 mV between 0 and +60 mV. Time constants measurements showed that NMG affected more the slow phase of inactivation of the Ca current. (5) When Ba was the charge carrier, NMG removed most of the inactivation of the current, suggesting a slowing of the voltage-dependent process of inactivation. (6) The results are consistent with a modification of the properties of the Ca channel by internal NMG.

Action Potentials↗

PubMed1988

"Run-down" of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium.

We examined by a statistical approach the decrease of the Ca current ("run-down") during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (ICa) occurs in three phases (T1-T3). T1 (38 +/- 19 min, n = 135) and T3 (35 +/- 17 min, n = 23) are characterized by a slow rate of decay of ICa [90 +/- 20 and 60 +/- 20 nA.cm-2.min-1, respectively]. T1 and T3 are separated by T2 (6 +/- 4 min, n = 135) during which the current decays quickly [1200 +/- 230 nA.cm-2.min-1]. Between the onsets of T1 and T3, ICa decreases from 11 +/- 3 to 3.5 +/- 1 microA/cm2. (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation of ICa are globally shifted toward more negative potentials during the run-down process by 10-15 mV. (3) ICa3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl2 and CoCl3, increase by isoprenaline and insensitivity to tetrodotoxin) of the original ICa. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down of ICa. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongs ICa survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors.

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.

Animals↗

PubMed1988

Receptor- and G-protein-mediated modulations of voltage-dependent calcium channels.

Explore the source record for details and available documents.

Animals↗

PubMed1988

Adrenoceptor-mediated changes of excitation and contraction in ventricular heart muscle from guinea-pigs and rabbits.

1. The influence of alpha-adrenoceptor stimulation on mechanical and electrophysiological parameters was investigated in ventricular preparations from guinea-pigs and rabbits. Action potential and force of contraction were measured in papillary muscles and ionic currents were measured in isolated myocytes. 2. The effects of alpha-adrenoceptor stimulation were compared with those of beta-adrenoceptor stimulation. 3. In the guinea-pig the stimulation of alpha-adrenoceptors caused a small increase in the force of contraction (less than 10% of the response to beta-adrenoceptor stimulation) which was not accompanied by any increase of the slow calcium inward current. beta-Adrenoceptor stimulation produced large increases in both force of contraction and slow inward calcium current. The noradrenaline-induced increase in the slow inward calcium current was not affected by phentolamine. 4. In the rabbit, alpha-adrenoceptor stimulation produced large increases in the force of contraction (about two thirds of those seen in response to beta-adrenoceptor stimulation). Whereas beta-adrenoceptor stimulation also produced large increases in both maximal upstroke velocity of slow-response action potentials and slow inward calcium current, there was almost no change of both parameters in response to alpha-adrenoceptor stimulation. 5. We conclude that, first, the contribution of alpha-adrenoceptors to adrenoceptor-mediated changes of force of contraction is minimal in the guinea-pig ventricle, and second, the pronounced changes of force of contraction in the rabbit ventricle in response to alpha-adrenoceptor stimulation are unrelated to changes in the slow inward calcium current.

Action Potentials↗