Phenylalkylamine Binding Sites Research Articles

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay.The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes.Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats.Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost.

Co-localization of putative calcium channels (phenylalkylamine-binding sites) on oil bodies in protoplasts from dark-grown sunflower seedling cotyledons

Oil bodies are spherical entities containing a triacylglycerol (TAG) matrix encased by a phospholipid monolayer, which is stabilized by oil body-specific proteins, principally oleosins. Biochemical investigations in the recent past have also demonstrated the expression of calcium-binding proteins, called caleosins, as a component of oil body membranes during seed germination. Using DM-Bodipy-phenylalkylamine (PAA; a fluorescent derivative of phenylalkylamine)-a fluorescent probe known to bind L-type calcium channel proteins, present investigations provide the first report on the localization and preferential accumulation of putative calcium channel proteins on/around oil bodies during peak lipolytic phase in protoplasts derived from dark-grown sunflower (Helianthus annuus L. cv Morden) seedling cotyledons. Specificity of DM-Bodipy-PAA labeling was confirmed by using bepridil, a non-fluorescent competitor of PAA while non-specific dye accumulation has been ruled out by using Bodipy-FL as control. Co-localization of fluorescence from DM-Bodipy-PAA binding sites (ex: 504 nm; em: 511 nm) and nile red fluorescing oil bodies (ex: 552 nm; em: 636 nm) has been undertaken by epifluorescence and confocal laser scanning microscopy (CLSM). It revealed the affinity of PAA-sensitive ion channels for the oil body surface. Findings from the current investigations highlight the significance of calcium and calcium channel proteins during oil body mobilization in sunflower.

Translocation of endothelial nitric oxide synthase: Another feat of amlodipine, a cardiovascular jack-of-all-trades

See article by Batova et al. [12] (pages 478–485) in this issue. The antihypertensive drug amlodipine (Norvasc®) is a long-acting, third-generation Ca2+ channel blocker that has been shown to limit the progression of atherosclerosis and to decrease the incidence of cardiovascular events in humans [1]. It is generally well tolerated and often used in combination with diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and statins [2–4]. Amlodipine exhibits nanomolar affinity to dihydropyridine as well as benzothiazepine and phenylalkylamine binding sites of voltage-dependent L-type Ca2+ channels [5]. It is several-fold more lipophilic than the first- and second-generation Ca2+ channel blockers and adopts a well-defined position in the bilayer membrane [6]. It has been suggested that partitioning of amlodipine to membranes results in beneficial modification of the structure of cholesterol-rich membranes of vascular smooth muscle cells, an effect that might contribute to atheroprotection [7]. Besides the well-documented inhibition of L-type Ca2+ channels, amlodipine exerts additional effects that contribute to the improvement of vascular function in hypertension and ischemic heart disease [8]. There is both in vitro and in vivo evidence indicating that dihydropyridines including amlodipine act as antioxidants preventing the progression of atherosclerosis by interfering with lipid peroxidation. However, amlodipine has pharmacological properties distinct from other Ca2+ channel blockers. Probably most relevant to the antihypertensive and antiplatelet effects of the drug is the release of endothelium-derived nitric oxide (NO). …

Investigation of the Phenylalkylamine Binding Site inhKv1.3 (H399T), a Mutant with a Reduced C-Type Inactivated State

To screen for residues of hKv1.3 important for current block by the phenylalkylamine verapamil, the inactivated-state-reduced H399T mutant was used as a background for mutagenesis studies. This approach was applied mainly to abolish the accumulation in the inactivated blocked state, recovery from which in the wild type is normally slow. Substitution of amino acids in the S6 transmembrane helix indicated a heavy disruption of verapamil block by the A413C mutation, reducing the IC(50) from 2.4 to 267 microM. Subsequent scanning for verapamil moieties essential for current block was performed by application of derivatives with altered side groups. Neither the removal of the nitrile or the methyl group nor the addition of a methoxy group resulted in major variations of IC(50) values for hKv1.3 (H399T) current block. However, disruption of current block by A413C was 4- to 10-fold less pronounced for derivatives lacking the 4-methoxy group of the (3,4-dimethoxyphenyl)ethylmethyl-amino part (devapamil) or all four methoxy groups (emopamil), respectively. Emopamil displayed a Hill coefficient of 2 for hKv1.3 (H399T/A413C) instead of 1 for hKv1.3 (H399T) current block. These results might indicate that the alteration of Ala413 modulates the access of phenylalkylamines to their binding site depending on the occupancy of the phenyl rings with methoxy groups. A computer-based docking model shows a subset of docked PAA conformations, with a spatial proximity between the (4-methoxyphenyl)ethyl-methyl-amino group and Ala413. The PAA binding site might therefore include a binding pocket for the aromatic ring of the ethyl-methyl-amino part in an S6-S6 interface gap.

Marrubenol interacts with the phenylalkylamine binding site of the L-type calcium channel

Marrubenol inhibits contraction of rat arteries by blocking L-type calcium (Ca2+) channels in smooth muscle cells, but its interaction with binding sites for calcium antagonists had never been investigated. Competition binding studies indicated that marrubenol was a weak inhibitor of 1,4-dihydropyridine binding in membranes isolated from rat intestinal smooth muscle but completely displaced specifically bound (−)-[3H]desmethoxyverapamil ([3H]D888) with an apparent Ki value of 16 μM (95% confidence interval: 6.5–39.5 μM). As marrubenol inhibited the contraction evoked by KCl depolarization of intestinal smooth muscle half-maximally at a concentration of ∼12 μM, interaction with the phenylalkylamine binding site seems to account for the inhibition of L-type Ca2+ channels by marrubenol.

Marrubenol interacts with the phenylalkylamine binding site of the L-type calcium channel

Marrubenol inhibits contraction of rat arteries by blocking L-type calcium (Ca 2+ ) channels in smooth muscle cells, but its interaction with binding sites for calcium antagonists had never been investigated. Competition binding studies indicated that marrubenol was a weak inhibitor of 1,4-dihydropyridine binding in membranes isolated from rat intestinal smooth muscle but completely displaced specifically bound (−)-[ 3 H]desmethoxyverapamil ([ 3 H]D888) with an apparent K i value of 16 μM (95% confidence interval: 6.5–39.5 μM). As marrubenol inhibited the contraction evoked by KCl depolarization of intestinal smooth muscle half-maximally at a concentration of ∼12 μM, interaction with the phenylalkylamine binding site seems to account for the inhibition of L-type Ca 2+ channels by marrubenol.

SR 33557, a novel calcium-antagonist: interaction with [3H]-(�)-nitrendipine and [3H]-(?)-desmethoxy-verapamil binding sites in cerebral membranes

We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC50 value of 5.6 +/- 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC50 = 96 +/- 22 nM). SR 33557 totally inhibited [3H]-(+/-)-nitrendipine binding to rat brain membranes with a Ki value of 0.19 +/- 0.03 nM. Diltiazem, which used alone increases [3H]-(+/-)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [3H]-(+/-)-nitrendipine binding. SR 33557 also fully inhibited [3H]-(-)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC50 value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC50 = 0.20 +/- 0.02 nM) was very similar to the Ki value determined for inhibition of [3H]-(+/-)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes.

Observation of polarity induction by cytochemical localization of phenylalkylamine-binding sites in regenerating protoplasts of the moss Physcomitrella patens.

Different external (e.g., light) and internal (e.g., auxin and calcium gradients) factors control differentiation of the moss protonema. The present investigations demonstrate that exogenously applied auxin, the pharmacological blockade of auxin efflux by naphthylphthalamic acid, and treatment with (-)bepridil, a calcium channel antagonist, inhibit protoplast division without affecting protoplast viability in the moss Physcomitrella patens. A fluorescently labelled phenylalkylamine (DM-Bodipy PAA), another calcium channel antagonist, was used as a probe for in vivo labelling of phenylalkylamine(PAA)-binding sites. The specificity of this binding was demonstrated by competition with (-)bepridil. Confocal laser scanning microscopy visualized PAA-binding sites on the plasma membrane and along the nuclear membrane as uniformly distributed clusters. During asymmetric division of P. patens protoplasts, however, fluorescence labelling particularly increases at the membrane invagination and later along the plate separating the new cells. Intracellular localization of PAA-binding sites, probably at the membranes of vesicles and vacuoles, significantly increases in the smaller daughter cell, destined to later form a polar outgrowth, the first chloronema cell. Thus, a system was established to visualize early events in P. patens protoplast polarization at the subcellular level.

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (−)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.

In vivo labeling of sunflower embryonic tissues by fluorescently labeled phenylalkylamine

A fluorescently labeled phenylalkylamine, DM-Bodipy PAA, was used as a probe for the in vivo detection of ion channels in embryonic and nonembryonic tissues of sunflower. Zygotic embryos, somatic embryos, callus, leaves, roots, and shoots were analysed. Fluorescence intensity in the tissues was determined with cytofluorometry and confocal microscopy. DM-Bodipy PAA intensively labeled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus cultures exhibited labeling on sites where somatic embryos developed. Labeling was, however, very weak in leaves, shoots, and roots, except in the root cap and in the epidermis of the root. Considering that the location of phenylalkylamine binding sites is related to the distribution of ion channels in both animal and plant cells, the high intensity of labeling observed in the protoderm and epidermis of zygotic and somatic embryos as well as in protoderm, epidermis, and caps of root tips, is consistent with the role these tissues may play in ion exchange with the environment.

Assessment of the antidepressant-like effects of L-type voltage-dependent channel modulators.

L-type voltage-dependent calcium channel blockers acting at different sites were tested in animal models of depression. Their effects on locomotion were studied in separate experiments. Nifedipine, a drug which interacts selectively with dihydropyridine (DHP) binding sites, reduced immobility time in the mouse forced swimming test and tail suspension test, but lacked activity in the differential-reinforcement-of-low-rate schedule in rats (DRL 72 s). The effects of nifedipine in the tail suspension test was partly antagonized by Bay K 8644, a DHP channel activator, indicating that its effect involved L-type calcium channel. Several other DHP drugs (nicardipine, nitrendipine, isradipine, felodipine and nimodipine) also showed antidepressant-like properties in the tail suspension test, whereas amlodipine, a less selective compound, lacked activity. In contrast to the DHP drugs, verapamil and (-)emopamil (which act at the phenylalkylamine binding sites), diltiazem and clentiazem (benzothiazepine binding sites), and the non-selective drug, flunarizine, were inactive in the tail suspension test. Negative results were also obtained with verapamil, diltiazem and flunarizine in the forced swimming test and with flunarizine on DRL 72 s responding. The present results show that DHP channel blockers displayed 'antidepressant-like' properties in mice. There was little dissociation, however, between the doses that produced antidepressant effects and those that decreased locomotor activity.

Molecular Determinants of High Affinity Phenylalkylamine Block of l-type Calcium Channels in Transmembrane Segment IIIS6 and the Pore Region of the α1Subunit

Recent studies of the phenylalkylamine binding site in the alpha1C subunit of L-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to L-type channels. We have extended this analysis of the phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the beta1b and alpha2delta auxiliary subunits. Assay for block of Ba2+ current by (-)-D888 at -60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between L-type and non-L-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one L-type-specific amino acid (Ile1153) decreased affinity for (-)-D888 from 10-20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (-)-D888 to approximately 9 microM, similar to the affinity of non-L-type Ca2+ channels for this drug. These results indicate that there are important determinants of phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor site at the interface between domains III and IV of the alpha1 subunit is presented.

1,5-benzothiazepine binding domain is located on the extracellular side of the cardiac L-type Ca2+ channel.

To determine whether 1,5-benzothiazepine Ca2+ channel blocker approaches its binding domain within the cardiac L-type Ca2+ channel from inside or outside of the membrane, we tested the effects of a novel potent 1,5-benzothiazepine derivative (DTZ323) and its quaternary ammonium derivative (DTZ417) on guinea pig ventricular myocytes by using the whole-cell patch-clamp technique. The extracellular application of DTZ417 suppressed the L-type Ca2+ channel currents (I[Ca(L)]) with an IC50 value of 1.2 +/- 0.02 microM, which was close to the IC50 value of diltiazem (0.63 +/- 0.01 microM). The suppression of I[Ca(L)] by DTZ417 was voltage and use dependent but lacked tonic block, which allowed us to investigate the onset of the effect on I[Ca(L)] by changing the holding potential (HP) from -90 to -50 mV in the presence of DTZ417. DTZ417 did not have significant effects on I[Ca(L)] at an HP of -90 mV. At -50 mV, DTZ417 (50 microM) applied from the extracellular side completely suppressed I[Ca(L)], whereas it had no effect from the intracellular side. DTZ323 (1 microM) also inhibited I[Ca(L)] only from the extracellular side, without any effects by the intracellular application of < or = 10 microM. However, a quaternary phenylalkylamine derivative, D890 (0.1 mM), acted only from the intracellular side. These results suggest that in contrast to the phenylalkylamine binding site, in cardiac myocytes the 1,5-benzothiazepine binding site is accessible from the extracellular side of the L-type Ca2+ channel.

Binding Sites for Ca2+‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

The calcium channel and the 'calcium release channel' of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0.1 and 300 μg ml -1 . Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [ 3 H]isradipine (PN 200-110), the phenylalkylamine [ 3 H]verapamil and the alkaloid [ 3 H]ryanodine. Preliminary binding studies with the benzothiazepine [ 3 H]diltiazem suggest a low-affinity binding site with a IC 50 value of 3.3 μM. All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM. EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca 2+ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca 2+ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca 2+ . Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K D values of 0.95 nM (B max = 550 fmol mg -1 protein) and 0.75 nM (B max = 213 fmol mg -1 protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K D = 7.4 nM and B max = 27 fmol mg -1 protein). [ 3 H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity : nitrendipine > isradipine > Bay K8664 >> nicardipine. Displacement of [ 3 H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.

Inhibitory effect of calcium channel blockers on human mesangial cell growth: Evidence for actions independent of L-type Ca2+ channels

Calcium channel blockers (CCB) are known to affect the outcome of glomerulosclerosis in vivo and to suppress mesangial cell proliferation and cytokine production in vitro. It is uncertain, however, whether (i) human adult mesangial cells (HMC) express L-type Ca2+ channels and (ii) whether the effect of CCB on HMC is mediated by inhibition of L-type Ca2+ channels. In single cell preparations of HMC, the L-type Ca2+ channel agonist Bay K 8644 and K+-depolarization of the cell membrane caused a transient increase of cytosolic free Ca2+ ([Ca2+]i) in 60 to 80% of the cells. The CCB verapamil and nifedipine partially inhibited the effect of Bay K 8644 and K+-depolarization on [Ca2+]i. Binding experiments confirmed these functional studies by showing specific binding at the phenylalkylamine binding site of L-Type Ca2+ channels. Quiescent HMC were stimulated with fetal calf serum (FCS) or growth factors (platelet derived growth factor A/B, epidermal growth factor, angiotensin II, endothelin 1) in the presence of various concentrations (10(-10) to 10(-5) M) of different CCB: either (R)-verapamil, (S)-verapamil or the raceme of verapamil, and nifedipine or diltiazem, respectively. In addition, the enantiomers of devapamil were studied, because their action on the L-type Ca2+ channel is more stereoselective than that of the enantiomers of verapamil. At high concentrations (10(-6) to 10(-5) M) (R,S)-verapamil decreased cell numbers in cultures of quiescent HMC, increased LDH in the supernatant, and caused loss of trypan blue exclusion (cytotoxicity). At lower concentrations (R,S)-verapamil showed no cytotoxicity, but had two effects: (1.) concentration dependent (down to 10(-8) M) inhibition of indices of cell proliferation, that is, (i) stimulated (FCS or growth factor) 3H-thymidine incorporation and (ii) increment in cell number; and (2.) inhibition of indices of cell or matrix protein synthesis, that is, (i) stimulated 3H-methionine incorporation and (ii) 3H-proline incorporation. At equimolar concentrations the dihydropyridine nifedipine was equipotent with verapamil, whereas the benzothiazepine diltiazem was conspicuously less effective. Even at the lowest effective concentration (10(-8) M) comparison of (R)- and (S)-verapamil showed no significant difference between the enantiomer with weak or with strong effect on L-type Ca2+ channels, and this was true even when the more stereoselective enantiomers of devapamil were tested. These observations argue against the notion that effects of CCB result from specific interaction with L-type Ca2+ channels. The data are more consistent with the idea that interactions with targets other than L-type Ca2+ channels are involved.

Effects of anandamide and arachidonic acid on specific binding of (+)-PN200-110, diltiazem and (−)-desmethoxyverapamil to L-type Ca 2+ channel

We examined the effects of anandamide ( N-arachidonoylethanolamine) on the binding of three types of Ca 2+ channel antagonists for L-type Ca 2+ channel, i.e., 1,4-dihydropyridine, 1,5-benzothiazepine and phenylalkylamine, to rabbit skeletal muscle membranes. Anandamide inhibited the binding of all three ligands. Arachidonic acid, a putative metabolite or a precursor of anandamide, inhibited 1,4-dihydropyridine binding, whereas it augmented both 1,5-benzothiazepine and phenylalkylamine binding. The involvement of prostaglandins synthesized from arachidonic acid was considered to be minor. These findings indicate that both anandamide and arachidonic acid interact not only with 1,4-dihydropyridine but also with 1,5-benzothiazepine and phenylalkylamine binding sites as a common feature of unsaturated lipids.

New purines and purine analogs as modulators of multidrug resistance.

A series of 36 purine and purine analog derivatives have been synthesized and tested for their ability to modulate multidrug resistance in vitro (P388/VCR-20 and KB-A1 cells) and in vivo (P388/VCR leukemia). Compounds were compared to S9788, a triazine derivative which has already shown some activity during phase 1 clinical trials and also a limiting cardiovascular side effect possibly linked to its calcium channel affinity. The fact that active compounds increase adriamycin accumulation in the resistant KB-A1 cells, and not in the sensitive KB-3-1 cells, suggests they act predominantly by inhibiting the P-glycoprotein-catalyzed efflux of cytotoxic agents. No direct relation was found between the affinity for the phenylalkylamine binding site of the calcium channel and in vitro sensitization of resistant cells. In vivo, when administered po in association with vincristine (0.25 mg/kg), five compounds (3, 4, 9, 25, and 26), of very differing calcium channel affinities (Ki from 5 to 560 nM), fully restored (T/V > or = 1.4) the sensitivity of P388/VCR leukemia to vincristine.

Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform

We report the complete sequence of a calcium channel alpha 1 subunit cDNA cloned from a Drosophila head cDNA library. This cDNA encodes a deduced protein containing 2516 amino acids with a predicted molecular weight of 276,493. The deduced protein shares many features with vertebrate homologs, including four repeat structures, each containing six transmembrane domains, a conserved ion selectivity filter region between transmembrane domains 5 and 6, and an EF hand in the carboxy tail. The Drosophila subunit has unusually long initial amino and terminal carboxy tails. The region corresponding to the last transmembrane domain (IVS6) and the adjacent cytoplasmic domain has been postulated to form a phenylalkylamine-binding site in vertebrate calcium channels. This region is conserved in the Drosophila sequence, while domains thought to be involved in dihydropyridine binding show numerous changes. The Drosophila subunit exhibits 78.3% sequence similarity to the rat brain type D calcium channel alpha 1 subunit, and so has been designated as a Drosophila melanogaster calcium channel alpha 1 type D subunit (Dmca1D). In situ hybridization shows that Dmca1D is highly expressed in the embryonic nervous system. Northern analysis shows that Dmca1D cDNA hybridizes to three size classes of mRNA (9.5, 10.2, and 12.5 kb) in heads, but only two classes (9.5 and 12.5 kb) in bodies and legs. PCR analysis suggests that the Dmca1D message undergoes alternative splicing with more heterogeneity appearing in head and embryonic extracts than in bodies and legs.

Molecular studies of the calcium antagonist binding site on calcium channels

Currently available calcium antagonists act primarily on L-type calcium channels. Composed of 5 subunits, this ion channel is markedly more complicated than the potassium and sodium channels. Most of the data that have emerged over the past year have concerned the α subunit. The secondary structure of this subunit includes 4 repeating motifs; each motif contains 6 membrane-spanning helices, designated S 1 through S 6. The dihydropyridine (e.g., nifedipine) binding site is most likely situated primarily in motif 4 and the phenylalkylamine (e.g., verapamil) binding site is on motif 4 in an intracellular region. The benzothiazepine (e.g., diltiazem) binding site is also located on the α 1 subunit, but its precise location has not been determined. The transmembrane helices S 5 and S 6 of each motif have been proposed as the “wall” of the actual ion channel.

Brain calcium channel related dihydropyridine and phenylalkylamine binding sites in Alzheimer's, Parkinson's and Huntington's diseases

Disruptions of normal calcium (Ca 2+) homeostasis in human aging process, are well documented. The existence of at least four distinct types of Ca 2+ channels, L-, T-, P-, and N-, have been reported in the centra l nervous system. Binding sites for clinically useful dihydropyridines (DPH) and phenylalkylamines (PA) are located on the L-type Ca 2+ channels. In the present study, DHP/[ 3H]PN200-110 and PA/[ 3H](−)-D-888 binding parameters were determined in various brain areas obtained at autopsy from Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) disease patients, and healthy age-matched controls as a means to assess the integrity of L-type channels in these neurological disorders often associated with the aging brain. DHP and PA receptor binding parameters ( K D and B max) were not significantly altered in any of the brain regions studied in AD and PD. However, a significant decrease in the maximal binding capacity of [ 3H]PN200-110 was observed in the striatum of HD patients. Taken together, this suggests that DHP and PA binding sites associated to L-type Ca 2+ channels are mostly preserved in AD and PD brains. Accordingly, the use of DHP- and/or PA-related drugs in these neurological disorders should not be hindered by deficits in their related Ca 2+ channel binding proteins.