Modulation Of Ca Channels Research Articles

Synthesis of carbon-11 labeled 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinolinium derivatives as new potential PET SK Ca channel imaging agents

Small conductance Ca 2+-activated K + (SK Ca) channels play an important role in many functions such as neuronal communication and behavioral plasticity, secretion, and cell proliferation. SK Ca channel modulation is associated with various brain, heart, and cancer diseases. N-methyl-laudanosine and its structurally related derivatives, substituted 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums, are reversible and selective SK Ca channel blockers. Carbon-11 labeled N-methyl-laudanosine and its tetrahydroisoquinolinium derivatives may serve as new probes for positron emission tomography (PET) to image SK Ca channels in the brain, heart, and cancer. The key intermediates, substituted isoquinolines ( 3a–c), were synthesized using a modification of the Pomeranz–Fritsch procedure. The precursors, substituted 1-(3,4-dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinolines ( 8a–c), and their corresponding reference standards, substituted 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums ( 9a–c), were synthesized from compounds 3a–c with 3,4-dimethoxybenzyl chloride ( 2) in multiple steps with moderate to excellent chemical yields. The precursor 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)-2-methyl-1,2,3,4-tetrahydroisoquinoline ( 10) was commercially available, and the methylation of compound 10 with methyl iodide provided N-methyl-laudanosine ( 11). The target quaternary ammonium tracers, carbon-11 labeled 1-(3,4-dimethoxybenzyl)-2,2-dimethyl-1,2,3,4-tetrahydroisoquinoliniums ([ 11C] 9a–c and [ 11C] 11), were prepared by N-[ 11C]methylation of the tertiary amine precursors ( 8a–c and 10) with [ 11C]methyl triflate and isolated by a simplified solid-phase extraction (SPE) purification using a SiO 2 or cation-exchange CM Sep-Pak cartridge in 40–65% radiochemical yields.

L-type calcium channel α-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current

Cardiac voltage-gated L-type Ca channels (Ca(V)) are multiprotein complexes, including accessory subunits such as Ca(V)beta2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel alpha-subunit (Ca(V)1.2) to Ca(V)beta2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies Ca(V)beta2-Rem inhibition of Ca channel current. We first tested the effect of Rem on Ca(V)1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with Ca(V)1.2 reduces Ba current expression under basal conditions, and Ca(V)beta2a coexpression enhances Rem block of Ca(V)1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 microM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without Ca(V)beta2a. To test whether the H-89 action was a consequence of the phosphorylation status of Ca(V)1.2, we examined Rem regulation of the PKA-insensitive Ca(V)1.2 serine 1928 (S1928) to alanine mutation (Ca(V)1.2-S1928A). Ca(V)1.2-S1928A current was not inhibited by Rem and when coexpression with Ca(V)beta2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the Ca(V)1.2 COOH terminus contributes to Rem-dependent channel inhibition.

Trapidil Enhances the Slowly Activating Delayed Rectifier Potassium Current and Suppresses the Transient Inward Current Induced by Catecholamine in Guinea Pig Ventricular Myocytes

We examined the electrophysiological effects of trapidil on the ionic currents influencing the repolarization and on the transient inward current (ITi) that can cause triggered arrhythmia using the whole-cell patch-clamp technique in guinea pig ventricular myocytes. Trapidil shortened the action potential duration (APD) and increased the delayed rectifier potassium current (IK) in a concentration-dependent manner. The effect of trapidil on the rapidly and slowly activating components of IK (IKr and IKs, respectively) was studied by the envelope of tails test. Trapidil failed to affect IKr and selectively enhanced IKs. Trapidil increased the amplitude of the L-type Ca2+ current (ICa,L), with an acceleration of its inactivation, whereas isoproterenol, a beta-adrenoceptor agonist, increased the amplitude of the ICa,L in a different manner. Isoproterenol activated ITi; however, trapidil not only failed to facilitate ITi but also suppressed isoproterenol-induced ITi. The inhibitory effect of trapidil on isoproterenol-induced ITi is at least partly via a reduction of Ca2+ overload through an acceleration of ICa,L inactivation and/or a sarcoplasmic reticulum (SR) Ca channel modulation. These results suggest that trapidil does not prolong the QT interval and has an antiarrhythmic effect on arrhythmias elicited by triggered activity secondary to Ca2+ overload at much higher concentrations than clinical concentration.

Interaction of BK Ca channel modulators with adrenergic agonists in the rat aorta is influenced by receptor reserve

Our main objective was to study the interaction of BK Ca channel modulators with adrenergic agonists UK 14304 and noradrenaline (NA), acting on α 1-adrenoceptors, in the rat aorta and how this is affected by receptor reserve. NA and UK 14304 evoked concentration-dependent contractions of the rat aorta. UK 14304 was a partial agonist relative to NA in this preparation. The BK Ca channel blocker tetraethylammonium (TEA, 1 mM) and opener NS 1619 (3×10 −5 M) modulated NA- and UK 14304-induced contractions, and were more effective on UK 14304-induced contractions. TEA (1 mM) increased the maximum response to NA and UK 14304 by about 13% and 300%, respectively, while NS 1619 (3×10 −5 M) reduced the maximum response to UK 14304 by about 81% compared to 31% for noradrenaline. The effect of TEA on the noradrenaline concentration–response curve was increased after treatment of the aorta with phenoxybenzamine (PBZ), an irreversible α 1-adrenoceptor antagonist, to reduce receptor reserve. We concluded that the interaction of BK Ca channel modulators with α 1-adrenergic agonists in the rat aorta was influenced by receptor reserve.

Potentiation of recombinant L-type Ca channel currents by α1-adrenoceptors coexpressed in baby hamster kidney (BHK) cells

In cardiac myocytes, the effect of α 1-adrenergic stimulation on L-type Ca current remains to be clarified. We examined this issue by the transient coexpression of α 1-adrenoceptors on BHKC12 cells, where recombinant Ca channels composed of cardiac α 1 subunit and skeletal β, γ, α 2 δ subunits were stably expressed. After transfection of plasmid DNA encoding bovine α 1 C -adrenoceptors, bath-applied phenylephrine potentiated the cloned Ca channel current during perforated-patch whole-cell recording by 26 ± 6 % in 6 out of 12 cells. The potentiation was elicited also by methoxamine, and was blocked by prazosin. Phenylephrine also increased the channel open probability during cell-attached single channel recording in 7 out of 15 cells. The ratio of successful modulation of Ca channels was in accordance with the ratio of successful expression of α 1-adrenoceptors, as estimated by β-galactosidase staining. These results suggest that the stimulation of α 1 C -adrenoceptors is linked to potentiation of cardiac L-type Ca current. BHK cells provide a valuable expression system to study the modulation of Ca channels evoked by a receptor stimulation.

Regulators of G protein signaling attenuate the G protein-mediated inhibition of N-type Ca channels.

Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Galpha, Gbetagamma, and their effectors. Voltage-gated N-type Ca channels mediate neurosecretion, and these Ca channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca channel function, we recorded the activity of N-type Ca channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuated the magnitude of receptor-mediated Ca channel inhibition. In control cells expressing alpha1B, alpha2, and beta3 Ca channel subunits and m2 receptors, carbachol (1 microM) inhibited whole-cell currents by approximately 80% compared with only approximately 55% inhibition in cells also expressing exogenous RGS protein. A similar effect was produced by expression of the conserved core domain of RGS8. The attenuation of Ca current inhibition resulted primarily from a shift in the steady state dose-response relationship to higher agonist concentrations, with the EC50 for carbachol inhibition being approximately 18 nM in control cells vs. approximately 150 nM in RGS-expressing cells. The kinetics of Ca channel inhibition were also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation was slower, and recovery of Ca channels from inhibition after agonist removal was faster than in control cells. The effects of RGS proteins on Ca channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Galpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca channels.

G-Protein-Dependent Facilitation of Neuronal α1A, α1B, and α1ECa Channels

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.

A new type of Ca-channel modulation by a novel class of 1,4-dihydropyridines.

Both Ca-antagonistic as well as Ca-agonistic 1,4-dihydropyridines (DHPs) have become extremely important tools to investigate the role of Ca-channels under various physiological and pathophysiological conditions. While Ca-antagonists stabilize the inactivated state of the Ca-channel without influencing the voltage dependent open and closed times, Ca-agonists prolong the mean open time of the channel. We here report for the first time the effects of a novel DHP, BAY Y 5959, which modulates Ca-channel gating in a unique manner: It increases both the mean open time and the mean closed time of the Ca-channel by binding to the DHP receptor. This results in a reduced rate of Ca-current activation, an increased peak current, and a strongly prolonged tail current decay. All these effects are strongly voltage dependent. Therefore it depends on resting membrane potential and shape of the action potential whether and how the Ca-influx into the cell is influenced. This novel mode of action of BAY Y 5959 results in an interesting pharmacological profile: It has a strong positive inotropic effect in the heart without influencing vessel tone. Therefore the term Ca-promoter is suggested; it could become a new approach for the drug treatment of congestive heart failure.

Interleukin‐lβ, tumor necrosis factor‐α, and LPS enhance calcium channel current in isolated vascidar smooth muscle cells of rat tail artery

Cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), are thought to be responsible for the compromised blood pressure regulation after systemic infection or other antigenic challenge. Because Ca homeostasis is critical for the maintenance of vascular tone, we hypothesized that cytokines may contribute to alterations in blood pressure by a mechanism involving the voltage-sensitive Ca channel in vascular smooth muscle (VSM) cells. Using nystatin-permeabilized patch techniques we examined the effects of IL-1 beta, TNF-alpha, and lipopolysaccharide (LPS) on the Ca channel of VSM cells isolated from rat tail artery. Both IL-1 beta (0.05--1 nM) and TNF-alpha (0.1--1 nM) increased, dose-dependently, the Ba2+ current carried in VSM Ca channels, whereas heat-denatured IL-1 beta was without significant effect on the channel. LPS (0.01--1.0 ng/ml) also increased the Ba2+ current with onset kinetics similar to the two cytokines. Prostaglandins were ruled out as an intermediary in VSM Ca channel modulation, as prostaglandin E2 had no effect and indomethacin (1 microM) failed to block TNF-alpha-induced Ca channel enhancement. The role of cyclic nucleotides in mediating TNF-alpha-induced changes in Ca channel activity was also assessed. Increasing intracellular cAMP via forskolin (1 microM) did not affect the response to TNF-alpha, but pretreatment with the membrane-permeant analog of cGMP, dibutyryl cGMP (100 microM), inhibited the response to TNF-alpha. These data demonstrate that IL-1 beta, TNF-alpha, and LPS have immediate effects on VSM cells via an interaction with the voltage-sensitive Ca channel, and these effects may he regulated by intracellular cGMP. Immunomodulation of Ca channels may represent an early signaling step in VSM cells mediating kinetically slower events, such as changes in gene transcription.

Electrodiffusive transport of Mg across renal membrane vesicles of the rainbow trout Oncorhynchus mykiss.

The mechanism of tubular Mg transport was investigated in membrane vesicles (MV) of trout kidneys prepared by differential centrifugation with sucrose. MV consisted largely of brush-border membranes, as indicated by high enrichments of brush-border membrane enzymes. Although measured transport of 28 Mg included a binding component, most membrane transport was into or out of an osmotically active space. There was no evidence for amiloride-sensitive Na/Mg exchange, nor was Mg uptake affected by the carboxyl group reagents trimethyloxonium tetrafluoroborate, glycine methyl ester.HCl-1-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide, and N,N'-dicyclohexyl carbodiimide or the Ca channel modulators D-600, verapamil, diltiazem, and BAY K 8644. However, Mg uptake increased in the presence of inside-negative voltages generated by inward gradients of the permeant anions NO3, SCN, and Cl or by outward gradients of K (plus valinomycin). Alkaline-earth cations displayed the selectivity sequence VII (Mg > Ca > Sr > Ba) for cis-inhibition of 28 Mg uptake. Mg efflux was trans-inhibited by La and Gd, and Mg uptake was cis-inhibited by Mn. The sulfhydryl group reagents p- chloromercuribenzoic acid and p-chloromercuriphenylsulfonate stimulated Mg uptake and efflux. These results reveal an electrodiffusive pathway for Mg transport in trout renal MV.

Signalling by cGMP-dependent protein kinases.

The second messenger cGMP is a major intracellular mediator of the vaso-active agents nitric oxide and natriuretic peptides. The principal targets of cGMP are (i) phosphodiesterases, resulting in interference with the cAMP-signalling pathway, (ii) cGMP-gated cation channels, and (iii) cGMP-dependent protein kinases (cGKs). Only two mammalian isotypes of cGK have been described so far: type I cGK, consisting of an alpha and a beta isoform, presumably splice variants of a single gene, and identified as the most prominent cGK isotype in the cardio-vascular system; and type II cGK, expressed mainly in the intestine, the kidney and the brain. High levels of cGK I are found in vascular smooth muscle cells, endothelial cells and platelets. In these cells, cGK I is thought to counteract the increase in contraction provoked by Ca-mobilizing agonists, to reduce endothelial permeability and to inhibit platelet aggregation, respectively. Relatively low levels of cGK I are found in cardiomyocytes. In this cell type, cGK is implicated in the negative inotropic effect of cGMP, presumably through modulation of Ca channels and by diminishing the Ca-sensitivity of contractile proteins.

Muscarinic inhibition of high-voltage-activated calcium channels in excised membranes of rat hippocampal neurons.

The effect of muscarine on voltage-gated calcium channels was investigated in outside-out patches from rat hippocampal neurons in culture. By clamping the excised patches at -60 mV holding potential, single and multiple Ca channel currents were recorded, and these displayed features similar to the high-voltage-activated Ca current, with unitary conductance of 6.4 pS in 50 mM external Ca2+. These channels turned out to be insensitive both to Bay K 8644 and to omega-conotoxin. In excised patches muscarine caused a marked reduction in the probability of opening of this class of Ca channels without significant changes in the unitary current amplitude. Interestingly, the degree of current inhibition was reduced by depolarization, thus suggesting a voltage-dependent inhibitory action of the agonist. We conclude that in hippocampal neurons one of the possible ways of HVA Ca channel modulation by muscarine occurs through activation of a substratum localized within the plasma membrane of the cell, independent of the involvement of diffusible intracellular messengers.

Characterization of single calcium channels in Drosophila embryonic nerve and muscle cells

Voltage-activated Ca channels play a central role in synaptic transmission, control of cell excitability, and many other cellular processes. It is now clear that there are multiple types of Ca channels with various modes of modulation. Drosophila offers exceptional advantages for studying the molecular basis of the diversity and modulation of Ca channels. As a step in this study, we have characterized the single-channel Ba currents recorded from cell-attached patches on cultured embryonic Drosophila nerve and muscle cells. The voltage dependence and selectivity of the channels carrying these Ba currents identify them as Ca channels. All Ca channels found in Drosophila neurons appear to have the same voltage dependence of activation and similar single-channel conductance, 12-17 pS (100 mM Ba2+). However, the kinetic properties of individual Ca channels vary greatly. The mean open time of individual channels ranges from 2 msec to less than 0.2 msec. Some channels completely inactivate during the first half of a 90 msec depolarization, while others are more active in the second half. Many channels open during almost every depolarization, while others open in less than 20% of the depolarizations. Channels with longer open times tend to inactivate and open during a small fraction of depolarizations. When these kinetic properties were quantified, a continuum of values was found, instead of the clustering of values that might be expected for discrete types of channels. Muscle Ca channels form a more homogeneous class than do the neuronal Ca channels. The muscle Ca channel conductance is 18 pS. These channels do not inactivate during 90 msec depolarizations and open during a majority of depolarizations applied. Muscle Ca channels are similar to a subset of neuronal Ca channels. When a purified toxin from the spider Hololena curta is applied to neurons, the number of active Ca channels is reduced, and those channels still active open in a small fraction of depolarization. Since channels that open in a small fraction of depolarizations tend to inactivate, these data support the hypothesis that this toxin selectively blocks noninactivating neuronal Ca channels. This differential toxin sensitivity and the much larger variability observed in kinetic properties of neuronal, compared to muscle, Ca channels suggest that there are at least two types of neuronal Ca channels in Drosophila.

Properties and modulation of Ca channels in adult human atrial cells

Ca-channel currents have been investigated in single cells isolated from adult human atrium using the whole-cell patch clamp technique. Ca-channel currents are activated at voltage positive to −40 mV, peak between −10 and 0 mV and inactivate with a slow decay when Ba 2+ ions (5 m m) are used as charges carrier. These properties correspond to those of the high voltage activated, DHP-sensitive, (L-type) Ca channel. No low voltage activated (T-type) currents have been evidenced. The present work also provides the first report about the modulation of Ca channels in adult human atrial cells by β-adrenergic agonists and dihydropyridines (agonists and antagonists). Electrophysiological and pharmacological properties of these Ca channels are qualitatively similar to those of the L-type Ca currents recorded from cardiac animal cells. However, at a physiological calcium concentration (2 m m), basal Ca currents are often very small or even absent but are revealed following the addition of the dihydropyridine (DHP) agonist Bay K 8644. Whether the decrease of the basal Ca current amplitude may be related to the chronic pretreatment of the patients by Ca channel blockers or to the pathology is discussed.

Modulation of the T-type cardiac Ca channel by changes in proton concentration.

Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.

Modulation of Ca Channels in Peripheral Neuronsa

Annals of the New York Academy of SciencesVolume 560, Issue 1 p. 346-357 Modulation of Ca Channels in Peripheral Neuronsa E. CARBONE, E. CARBONE Dipartimento di Anatomia e Fisiologia Umana Corso Raffaello 30 I-10125 Torino, ItalySearch for more papers by this authorH. D. LUX, H. D. LUX Max-Planck-Institute fur Psychiatrie Am Klopferspitz 18A D-8033 Planegg, Federal Republic of GermanySearch for more papers by this author E. CARBONE, E. CARBONE Dipartimento di Anatomia e Fisiologia Umana Corso Raffaello 30 I-10125 Torino, ItalySearch for more papers by this authorH. D. LUX, H. D. LUX Max-Planck-Institute fur Psychiatrie Am Klopferspitz 18A D-8033 Planegg, Federal Republic of GermanySearch for more papers by this author First published: June 1989 https://doi.org/10.1111/j.1749-6632.1989.tb24114.xCitations: 4 a This work was partially supported by NATO (Grant No. 576187) and the Consiglio Nazionale delle Ricerche (Grant No. 88.00458.04). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume560, Issue1Calcium Channels: Structure and FunctionJune 1989Pages 346-357 RelatedInformation

Calcium channel modulation by the dihydropyridine derivative bay K 8644 in mammalian visceral smooth muscle cells

1. 1. Modulation of Ca channels by the dihydropyridine Ca agonist Bay K 8644 in guinea-pig taenia coli smooth muscle cells was investigated using the patch clamp technique. 2. 2. Single Ca channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 50 mM Ba. Bath application of the drug markedly increased the opening probability of Ca channels. 3. 3. The effect was found to be due to an increase in the mean opening times of Ca channels. Due to this increase, the mean current reconstructed by averaging individual current trace responses was markedly increased in the presence of Bay K 8644.

Modulation of Ca channels in cardiac muscle

Modulation of Ca channels in cardiac muscle

Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644.

A large-conductance calcium channel in chicken dorsal root ganglion neurons was studied with patch-clamp recordings of unitary currents. In addition to the conventional pattern of Ca-channel gating previously described in neurons ("mode 1"), we observed a different form of gating behavior ("mode 2"). Unlike the brief (approximately equal to 1 ms) openings in mode 1, mode 2 openings tend to be longer (greater than 10 ms) and often outlast the test pulse. In mode 2, the probability of channel openness (P) is high at relatively negative potentials where P in mode 1 is low. Mode 2 activity appears much less often than mode 1 activity in the absence of drug. However, the balance is strongly shifted in favor of mode 2 by the dihydropyridine Ca agonist Bay K 8644, an effect that underlies a marked enhancement of Ca-channel activity. This is the first evidence for dihydropyridine control of neuronal Ca-channel function at the single-channel level. Sweeps showing mode 1 or mode 2 gating appeared interspersed with sweeps with no openings, during which the channel was unavailable for opening ("null mode" or "mode 0"). Two approaches showed that switching between all three modes occurred on a time scale of seconds: (i) channels tended to remain in the same mode from one sweep to the next, with pulses at 0.25 Hz; and (ii) steady depolarizations in Bay K 8644 produced clusters of mode 2 openings lasting several seconds. Changes in the rates of switching might be important in neurochemical modulation of Ca channels. Bay K 8644 and other dihydropyridine Ca agonists might be useful experimental tools for manipulating transmitter release, neurite extension, and other neuronal functions dependent on intracellular Ca.