Heterogeneity of calcium channel agonist binding sites in the coronary artery

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.     

Activation of a covalent outer membrane phospholipase A dimer.

The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.     

Molecular Pharmacology of High Voltage-Activated Calcium Channels

Voltage-gated calcium channels are key sources of calcium entry into the cytosol of many excitable tissues. A number of different types of calcium channels have been identified and shown to mediate specialized cellular functions. Because of their fundamental nature, they are important targets for therapeutic intervention in disorders such as hypertension, pain, stroke, and epilepsy. Calcium channel antagonists fall into one of the following three groups: small inorganic ions, large peptide blockers, and small organic molecules. Inorganic ions nonselectively inhibit calcium entry by physical pore occlusion and are of little therapeutic value. Calcium-channel-blocking peptides isolated from various predatory animals such as spiders and cone snails are often highly selective blockers of individual types of calcium channels, either by preventing calcium flux through the pore or by antagonizing channel activation. There are many structure-activity-relation classes of small organic molecules that interact with various sites on the calcium channel protein, with actions ranging from selective high affinity block to relatively nondiscriminatory action on multiple calcium channel isoforms. Detailed interactions with the calcium channel protein are well understood for the dihydropyridine and phenylalkylamine drug classes, whereas we are only beginning to understand the molecular actions of some of the more recently discovered calcium channel blockers. Here, we provide a comprehensive review of pharmacology of high voltage-activated calcium channels.     

Molecular tools for the study of calcium channels: synthesis and evaluation of biological activity of new derivatives of 4-aryl-1,4-dihydropyridine groups

The synthesis and biological activity of some novel analogs of the calcium channel blocker nifedipine, i.e., derivatives of 2.6-dimethyl-3.5-diethoxycarbonyl-4-(3-nitrophenyl)-1.4-dihydropyridine (DHP) were studied. One radioactive and two photoactivable DHP derivatives were obtained. DHP hemisuccinate was used to prepare an affinity matrix, DHP-Sepharose as well as a DHP-albumin conjugate; the latter was used for anti-DHP antibodies generation in rabbits. All novel DHP derivatives were obtained from a single key 3-hydroxycarbonyl DHP derivative, and they comprise a series of necessary tools for the study and isolation of membrane calcium channels.     

Discovery of Michael acceptor containing 1,4-dihydropyridines as first covalent inhibitors of L-/T-type calcium channels

1,4-Dihydropyridines (DHPs) are an important class of blockers targeting different calcium channel subtypes and have great therapeutic value against cardiovascular and neurophysiologic conditions. Here, we present the design of DHP-based hexahydroquinoline derivatives as either selective or covalent inhibitors of calcium channels. These compounds were synthesized via a modified Hantzsch reaction under microwave irradiation and characterized by IR, ¹H NMR, ¹³C NMR and mass spectra. Additionally, the proposed structure of HM12 was resolved by single crystal X-ray analysis. The abilities of the target compounds to block both L- and T-type calcium channels were evaluated by utilizing the whole-cell patch clamp technique. Our results identified covalent inhibitors of calcium channels for the first time, which could be achieved by introducing a Michael acceptor group into the ester side chain of the compounds. The proposed covalent binding between the compounds and the cysteine amino acid (Cys1492) within the DHP binding pocket of L-type calcium channel was supported by docking and pharmacophore analysis as well as a glutathione reactivity assay.     

Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effect of modulators

1. Loss of response after prolonged or repeated application of stimulus is generally termed desensitization. A wide variety of phenomena occurring in living organisms falls under this general definition of desensitization. There are two main types of desensitization processes: specific and non-specific. 2. Desensitization of the nicotinic acetylcholine receptor is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. It is a case of specific desensitization and is an intrinsic molecular property of the receptor. 3. Desensitization of the nicotinic acetylcholine receptor at the neuromuscular junction was first reported by Katz and Thesleff in 1957. Desensitization of the receptor has been demonstrated by rapid kinetic techniques and also by the characteristic "burst kinetics" obtained from single-channel recordings of receptor activity in native as well as in reconstituted membranes. In spite of a number of studies, the detailed molecular mechanism of the nicotinic acetylcholine receptor desensitization is not known with certainty. The progress of desensitization is accompanied by an increase in affinity of the receptor for its agonist. This change in affinity is attributed to a conformational change of the receptor, as detected by spectroscopic and kinetic studies. A four-state general model is consistent with the major experimental observations. 4. Desensitization of the nicotinic acetylcholine receptor can be potentially modulated by exogenous and endogenous substances and by covalent modifications of the receptor structure. Modulators include the noncompetitive blockers, calcium, the thymic hormone peptides (thymopoietin and thymopentin), substance P, the calcitonin gene-related peptide, and receptor phosphorylation. Phosphorylation is an important posttranslational covalent modification that is correlated with the regulation and desensitization of the receptor through various protein kinases. 5. Although the physiological significance of desensitization of the nicotinic receptor is not yet fully understood, desensitization of receptors probably plays a significant role in the operation of the neuronal networks associated in memory and learning processes. Desensitization of the nicotinic receptor could also possibly be related to the neuromuscular disease, myasthenia gravis.     

Molecular determinants of syntaxin 1 modulation of N-type calcium channels

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939-2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein beta subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to Gbeta and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH(2)-terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with Ggamma for the Gbeta subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein gamma subunit-like domain.     

Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.     

A method for estimation of drug affinity constants to the open conformational state of calcium channels.

The affinity of D600 to calcium channels in the open state has been examined in isolated smooth muscle cells of the rabbit ear artery. Calcium channel currents were measured in high external barium solution by means of the patch-clamp technique. The current inhibition in various D600 concentrations (3-100 microM) on application of trains of short test pulses (20-80 ms) has been studied in nonmodified calcium channels and in cells where the calcium channels were modified by the agonist dihydropyridine (+) 202,791 (100 nM). The kinetics of the peak current decay has been analyzed with a mathematical model which is based on the experimental finding that D600 interacts primarily with calcium channels in the open conformational state. The model approach allows the estimation of drug affinity constants of D600 to the calcium channel in the open conformation. An association rate constant to the open conformational state of D600 of 6.16 x 10(4) M-1 s-1 was estimated. The association rate of the drug was not significantly changed after the calcium channels have been modified with 100 nM (+) 202,791. A method for correction of rate constants for possible drug trapping is discussed.     

Nitrendipine and isoproterenol induce phosphorylation of a 42,000 dalton protein that co-migrates with the affinity labeled calcium channel regulatory subunit

Slow inward calcium channels in canine cardiac membranes were affinity labeled with the calcium channel analogue, [3H]o-NCS [2,6 dimethyl-3,5-dicarbomethoxy-4-(2- isothiocyanatophenyl )-1, 4-dihydropyridine], in the presence and absence of cold o-NCS or nicardipine. A major specifically labeled peak was identified with Mr 42,000 on NaDodSO4 polyacrylamide gels. In parallel experiments the effects of the calcium channel antagonist, nitrendipine and a variety of other chemical mediators were tested for their ability to stimulate protein phosphorylation in cardiac membranes. These data demonstrate that both nitrendipine and isoproterenol induce the phosphorylation of a 42,000 dalton protein via a kinase endogenous to the cardiac membranes and that the effects of isoproterenol are attenuated by carbachol.     

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.     

Platelet-derived growth factor activation of gastric mucosal calcium channels.

Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhancement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Low voltage activated calcium channels: from genes to function

Cloning of three members of low-voltage-activated (LVA) calcium channel family, predominantly neuronal alpha1G and alpha1I, and ubiquitous alpha1H, enabled to investigate directly their electrophysiological and pharmacological profile as well as their putative subunit composition. All the three channels are half-activated at membrane potential about -40 mV and half-inactivated at about -70 mV. Kinetics of alpha1G and alpha1H channels activation and inactivation are similar and faster than that of alpha1I channel. All the three channels are blocked with high affinity by the organic blocker mibefradil. Another high affinity blocker is kurtoxin. Cloned LVA channels are relatively insensitive to antiepileptics, dihydropyridines and omega-conotoxins. Ni2+ is high affinity blocker of alpha1H channel only. Amiloride inhibits the alpha1H channel. The subunit composition of LVA channel remains unclear. Out of known high-voltage-activated calcium channel subunits, alpha2delta-2 and gamma-5 subunits significantly and systematically modified activation and/or inactivation of the current. In contrast, alpha2delta-1, alpha2delta-3, gamma-2 and gamma-4 subunits failed to modulate the current or had only minor effects.     

Modulation of gastric mucosal calcium channels activity by platelet-derived growth factor.

A dihydropyridine-sensitive gastric mucosal calcium channels were isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The channels following labeling the calcium antagonist receptor site with [3H]PN200-100 were reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake as evidenced by La3+ displacement assays. The uptake of calcium was independent of sodium and potassium gradients indicating the electroneutral nature of the process. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microns exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhacement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channel protein showed an increase in tyrosine phosphorylation of 55 and 170 kDa calcium channel proteins. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results demonstrate the importance of PDGF in the regulation of gastric mucosal calcium uptake.     

容量性钙内流通道的分子代表

细胞内钙库排空产生一种信号,诱导细胞膜上的钙库操纵的钙通道（SOC）开放,使Ca^2 由细胞外进入细胞内,称为容量性钙内流（CCE）,或钙释放激活的钙通道（CRAC）,可能由果蝇一过性受体电位（trp)和trp样（trpl)基因编码,钙库排空和通道开放之间的偶联机制不清,目前主要提出三种机制：（1）弥散信使;（2）蛋白质－蛋白质之间的相互作用;（3）囊泡分泌。本文综述了CCE的分子代表 ,可能机制及电生理表型。     

The effects of chronic depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent Ca2+ channels in chick neural retina and rat cardiac cells.

Chick neural retina cells contain functional L-type voltage-dependent Ca2+ channels sensitive to 1,4-dihydropyridines. To investigate the effects of chronic depolarization, cells were grown in medium containing elevated K+. After 4-h to 4-day treatments with elevated K+ (12-73 mM), there was a concentration-dependent decrease in high affinity [3H]PN200-110 binding. Saturation analysis of cells treated for 4 days with 40 mM K+ showed a reduction in maximum ligand binding with no change in affinity. Control and experimental Bmax values were 70.7 +/- 6.4 and 42.2 +/- 4.5 fmol/mg protein, respectively, and control and experimental KD values were 70.2 +/- 7.4 and 68.6 +/- 7.4 x 10(-12) M. The effect of chronic depolarization was time-dependent, reversible, and without effect on cellular protein content. Reduction in 45Ca2+ uptake following chronic depolarization correlated well with the reduction in [3H]PN200-110 binding. The calcium ionophore A23187, 10(-6) M for 24 h, also decreased the binding site density. The calcium channel antagonist D600 had no effect alone on [3H]PN200-110 binding; however, D600 blocked the down-regulation of calcium channels induced by chronic depolarization. The mechanism for Ca2+ channel down-regulation may involve calcium entry, since the effect was blocked by D600 and mimicked by the calcium ionophore A23187. Chronic depolarization with either elevated K+ or veratridine, or chronic treatment with A23187 had no effect on calcium channels in rat neonatal ventricular myocytes, although these cells express functional channels of the 1,4-dihydropyridine-sensitive class.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and characterization of a novel calcium channel from rabbit brain.

The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L-type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum.     

Modulation of dihydropyridine-sensitive gastric mucosal calcium channels by GM1-ganglioside.

1. A dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal cell membranes and purified by affinity chromatography on wheat germ agglutinin. 2. The calcium channel complex labeled with [3H]PN200-110, when reconstituted into phosphatidylcholine vesicles, exhibited active 45Ca2+ uptake into intravesicular space as evidenced by La3+ displacement and osmolarity studies. The channel complex responded in a dose-dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory effect of 66% in 45Ca2+ uptake. 3. The uptake of 45Ca2+ into vesicle-reconstituted gastric mucosal calcium channel complex was inhibited by GM1-ganglioside. Maximum inhibitory effect was achieved at 10-15 nM GM1, at which point a 74% decrease in 45Ca2+ uptake occurred. Furthermore, GM1 also inhibited dihydropyridine binding to gastric mucosal membranes, indicating the extracellular orientation of calcium channel domains for GM1. 4. The ability of GM1 to modulate the intracellular calcium levels may be an important feature in gastric mucosal protection by this ganglioside.