Drug Receptor Sites Research Articles

An innovative and universal dual-signal ratiometric spectro-electrochemical imprinted sensor design for sandwich type detection of anticancer-drug, gemcitabine, in serum samples; cross validation via computational modeling

An innovative and universal imprinted sensor design for sandwich type detection of gemcitabine (GMT) in human serum samples is described. GMT is widely used in the treatment of different tumors, such as lung, ovarian, pancreatic, and breast cancer. The serum albumin-drug interaction was translated to design a multifunctional, ratiometric and dual mode silver nanoparticle based probe (BSA-Ag nanoprobe), as a read out system. Subsequently, polypyrrol imprinted drug receptor sites was engineered to selectively capture the GMT on the transducer surface. The GMT was sandwiched between imprinted receptor surface and BSA-Ag nanoprobe to generate the spectro-electrochemical signals. The formation of nanoprobe was confirmed through various characterization techniques, including X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, micro-Raman spectroscopy, Dynamic light scattering (DLS), and UV–Visible (UV–Vis) analysis, while each step of sensor fabrication was characterized via field emission scanning electron microscope (FE-SEM), Static water Contact angle measurements, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Different variable parameters were optimized to improve the analytical performance of the sensor design. Under optimal conditions, spectro-electrochemical sensor permitted linear ranges between 1 and 200 μmol L−1 and 0.5–200 μmol L−1, with limits of detection (LOD) of 0.4 μmol L−1 and 0.15 μmol L−1 respectively. Furthermore, the designed sensor successfully differentiated the serum samples of lung cancer patients and healthy volunteers. The obtained results were validated with standard Liquid chromatography-mass spectrometry (LC/MS) analysis of the patients and healthy volunteer's serum samples. Lastly, density functional theory (DFT) and molecular docking calculations revealed the enhanced GMT binding capability of molecularly imprinted polypyrrole and molecular level interaction between the GMT and BSA, to validate the sandwich sensor design.

Druggability of Voltage-Gated Sodium Channels-Exploring Old and New Drug Receptor Sites.

Voltage-gated ion channels are important drug targets because they play crucial physiological roles in both excitable and non-excitable cells. About 15% of clinical drugs used for treating human diseases target ion channels. However, most of these drugs do not provide sufficient specificity to a single subtype of the channels and their off-target side effects can be serious and sometimes fatal. Recent advancements in imaging techniques have enabled us for the first time to visualize unique and hidden parts of voltage-gated sodium channels in different structural conformations, and to develop drugs that further target a selected functional state in each channel subtype with the potential for high precision and low toxicity. In this review we describe the druggability of voltage-gated sodium channels in distinct functional states, which could potentially be used to selectively target the channels. We review classical drug receptors in the channels that have recently been structurally characterized by cryo-electron microscopy with natural neurotoxins and clinical drugs. We further examine recent drug discoveries for voltage-gated sodium channels and discuss opportunities to use distinct, state-dependent receptor sites in the voltage sensors as unique drug targets. Finally, we explore potential new receptor sites that are currently unknown for sodium channels but may be valuable for future drug discovery. The advancement presented here will help pave the way for drug development that selectively targets voltage-gated sodium channels.

Anthelmintic Resistance and Its Mechanism: A Review.

Helminths are a various types of parasites causing a major health problem for animals in different parts of the globe. Control of helminthiasis has largely relied on the use of pharmaceutical anthelmintics. Unfortunately, the exhaustive use of anthelmintic drugs has led to a serious and dramatic level of anthelmintic resistance. Anthelmintic resistance is a heritable loss of sensitivity of an anthelmintic in a parasite population that was in the past susceptible to the same anthelmintic. The development of anthelmintic resistance is evident to different helminths of almost every animal species and to different groups of anthelmintic in several continents. Frequent treatment, underdosing, genetics of the parasite, and targeting and timing of mass treatment are predisposing factors for anthelmintic resistance. Upregulation of cellular efflux mechanisms, an increase in drug metabolism, a change in drug receptor sites that reduces drug binding or the functional consequences of drug binding, and a decrease in drug receptor abundance through reduced expression within the parasite are the main mechanisms of anthelmintic resistance. In vivo method like fecal egg count reduction test and in vitro method such as egg hatch assays, larval motility test, larval development test and PCR can be used for the detection of anthelmintic resistance. Proper utilization of anthelmintic drugs, using combined anthelmintic and applying other alternatives are essential strategies to slow down the development of anthelmintic resistance. As anthelmintic resistance is a serious challenge throughout the world, proper utilization of the existing anthelmintics and reducing dependence on anthelmintics should be implemented to reduce its challenge.

Site occupancy calibration of taxane pharmacology in live cells and tissues

Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.

Functional and Molecular Interaction of Phenylalkylamines and Dihydropyridines with the Model Calcium Channel CavAb

Mammalian voltage-gated calcium channels (mCavs) are molecular targets of phenylalkylamines (PAAs) and dihydropyridines (DHPs), which are widely used antiarrhythmic and antihypertension drugs that block mCavs in a frequency- and voltage-dependent manner. The two distinct receptor sites for these drugs in mCavs have been elucidated through extensive photoaffinity labeling and mutagenesis studies, revealing key residues along the S5 and S6 transmembrane segments of domains III and IV of mCavs (Hockerman et al., 1997). We have recently reported the crystal structure of CavAb, which is a highly calcium-selective mutant of the prokaryotic sodium channel NavAb (Tang et al., 2014). Even though CavAb is separated evolutionarily from mCavs by more than one billion years, we found that PAAs and DHPs interact with CavAb in a similar state-dependent manner. The PAAs tested have Kd's in the range of 250 nM to 800 nM, and verapamil shows low-affinity block of the resting state and high-affinity use-dependent block of activated and/or inactivated states. Mutations of T206 in the center of segment S6 reduced PAA affinity by 20-fold. In the case of DHPs, nimodipine showed voltage-dependent block of activated CavAb with a Kd of 194 nM, but no resting-state block was observed in the tested concentration range. DHPs shift the voltage dependence of inactivation up to 100 mV negatively. This indicates that DHP interaction with CavAb is state-dependent and greatly favors the inactivated state, similar to mCavs. Mutation of I199 in the outer region of segment S6, which is critical for binding DHPs in mCavs, reduced binding affinity 40-fold. Our results reveal surprising similarity of drug action on CavAb and mCavs and thereby support use of CavAb as a molecular model to determine the three-dimensional structure of these important drug receptor sites.

Indoxacarb, metaflumizone, and other sodium channel inhibitor insecticides: Mechanism and site of action on mammalian voltage-gated sodium channels

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (e.g., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the “local anesthetic (LA) receptor.” This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.

Drug - Receptor Interactions

In present-day pharmacology and medicine, it is usually taken for granted that cells contain a host of highly specific receptors. Drugs act on the cell membrane by physical and/or chemical interactions. This is usually through specific drug receptor sites known to be located on the membrane. These are defined as proteins on or within the cell that bind with specificity to particular drugs, chemical messenger substances or hormones and mediate their effects on the body. Today, the concept of specific receptors for drugs and transmitters lies at the very heart of pharmacology. However, it is only relatively recently that the notion of drug-specific receptors has become widely accepted, with considerable doubts being expressed about their existence as late as the 1960s.

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states.

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

DNA Topoisomerase II Structures and Anthracycline Activity: Insights into Ternary Complex Formation

DNA Topoisomerase II (Top2) is an essential nuclear enzyme that regulates the topological state of the DNA, and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have revised the published crystal structures of eukaryotic and prokaryotic Top2s and relevant biochemical investigations of enzyme activity and anthracycline action. In particular, we have considered Top2 mutations conferring resistance to anthracyclines and related agents. Following a previous study (Moro et al, Biochemistry, 2004; 43: 7503-13), we have then re-built a molecular model of the entire enzyme in complex with DNA after the cleavage reaction, and used it to define the receptor site of anthracyclines. The results suggest a model wherein the drug specifically contacts the cleaved DNA as well as amino acid residues of the enzyme CAP-like domain. The findings can explain several established structure-activity relationships of antitumour anthracyclines, and provide a framework for further developments of effective Top2 poison.

Interaction model for anthracycline activity against DNA topoisomerase II.

DNA topoisomerase II (Top2) is an essential nuclear enzyme and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have investigated the sensitivity to three closely related anthracycline derivatives of yeast Top2 bearing mutations in the CAP-like domain and integrated the findings with computer models of ternary drug-enzyme-DNA complexes. The results suggest a model for the anthracycline receptor wherein a drug molecule has specific interactions with the cleaved DNA as well as amino acid residues of the CAP-like domain of an enzyme monomer. The drug molecule is intercalated into DNA at the site of cleavage, and interestingly, drug-enzyme contacts involve one side of the four-ring chromophore and the side chain of the anthracycline molecule. The findings may explain several established structure-activity relationships of antitumor anthracyclines and may thus provide a framework for further developments of effective Top2 poisons.

Befloxatone, a new reversible and selective monoamine oxidase-A inhibitor. I. Biochemical profile.

Befloxatone, a novel oxazolidinone derivative, inhibited selectively and competitively monoamine oxidase (MAO)-A in human and rat brain, heart, liver and duodenum homogenates with Ki values ranging from 1.9 to 3.6 nM for MAO-A and from 270 to 900 nM for MAO-B. In vitro, befloxatone was more potent at inhibiting MAO-A activity than reference compounds (befloxatone > harmaline > brofaromine > BW 137OU87 > RS 8359 > toloxatone > moclobemide). The inhibition of MAO-A by befloxatone was time-dependent and fully reversible after dilution. After p.o. administration, befloxatone induced a dose-dependent and selective inhibition of rat brain and duodenum MAO-A activities ex vivo with ED50 values of 0.06 and 0.025 mg/kg, respectively. Befloxatone (0.5 mg/kg p.o.) decreased MAO-B activity by only 20% in both tissues. In the brain, liver and duodenum, the inhibition of MAO-A activity by befloxatone was short lasting. Twenty-four hours after administration of befloxatone (0.75 mg/kg p.o.), a full recovery of MAO-A activity was observed in the brain, but the enzyme activity was still decreased by 38 and 56% in the duodenum and liver, respectively. In the rat brain, befloxatone (0.75 mg/kg p.o.) increased levels of norepinephrine, dopamine and 5-hydroxytryptamine and decreased levels of their respective deaminated metabolites. These variations were dose-dependent and reversed 24 hr after administration. In addition, befloxatone (0.75 mg/kg p.o.) decreased free 3,4-dihydroxyphenylethylene glycol levels in the brain and plasma. Befloxatone (10 microM) did not modify the activities of diamine or benzylamine oxidase and did not interact with monoamine uptake mechanisms or with a variety of neurotransmitter or drug receptor sites. In conclusion, the neurochemical profile of befloxatone demonstrates that this compound is a selective, competitive, potent and reversible MAO-A inhibitor.

Chemotherapy of Nematode Infections of Veterinary Importance, with Special Reference to Drug Resistance

Publisher Summary The historical succession of one drug class after another has resulted in part from sequential improvement in drug characteristics (spectrum, safety, convenience), but also in part from the emergence of drug resistance. The phenomenon of resistance, so characteristic of the treatment of infectious diseases in general, is a prominent feature of the new era in antinematodal therapy. Anthelmintic use may vary to some extent in various parts of the world, but it rests largely on a common set of chemical compounds. An aspect of the older drugs is the development of new delivery technology. Administration of the injectable product gives high efficacy against many nematode and arthropod parasites, and these are disucssed in the chapter. The drug is active against gastrointestinal and pulmonary nematodes, against immature as well as mature worms, and against inhibited fourth-stage larvae. The susceptible arthropods include ticks, lice, mites, and grubs. Drug resistance is a heritable reduction in the sensitivity of a parasite population to the action of a drug, the reduction being expressed as a decrease in the frequency of individual parasites affected by exposure to the drug. Drug resistance generally results from decreased drug uptake, increased metabolism of the drug, or changes at the drug-receptor site.

Voltage-dependent calcium channels: structures and drug-binding sites.

I47 Tsien’s group classified these channels as ‘N, ‘T, In the 1960s the research groups of Fleckenstein and Godfraind defined ‘calcium antagonists’ as agents which blocked the entry of calcium into cells via voltage-dependent calcium channels. In a literature search we found 74 compounds in various stages of clinical or preclinical development classed as ‘calcium antagonists’. Since the initial work of Fleckenstein [ l ] and Godfraind et al. [2], it has become apparent that there are many different types of calcium channel, that there are different binding sites for drugs on the various channels, and that the binding of some of the drugs is highly voltage-dependent, allowing selective interactions with certain states of the channels and selective therapeutic interventions. The application of molecular biology has resulted in the definition of the primary structure of many subtypes of the calcium channel and the first steps are being made to define the structure of the drug receptor sites on the channel proteins.

Acceleration of activation and inactivation by the β subunit of the skeletal muscle calcium channel

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.

THE ROLE OF CALCIUM IN Chlamydomonas PHOTOMOVEMENT RESPONSES AS ANALYSED BY CALCIUM CHANNEL INHIBITORS

Phototaxis and light-induced stop responses in Chlamydomonas are known to be calcium dependent. We show that phototaxis is stereoselectively inhibited by dihyropyridines, verapamil, diltiazem, omega-conotoxin and pimozide, all inhibitors of slow L-type calcium channels. In contrast, the stop response in Chlamydomonas can be specifically reduced only by omega-conotoxin and pimozide. The light-regulated calcium uptake as detected by 45calcium can be completely suppressed by verapamil and omega-conotoxin but not by diltiazem or any of the dihyropyridine-type calcium channel inhibitors. We conclude that phototaxis and stop response in Chlamydomonas are regulated by three distinguishable drug receptor sites. One of them controls phototaxis and is sensitive to verapamil. The second site controls stop response and phototaxis and shows a high sensitivity to omega-conotoxin and pimozide. These two drug receptors seem to be localized in the plasma membrane and function as ion channels. In addition, calcium influences internal signal transduction from the photoreceptor to the flagella. This internal role of calcium is inhibited by the dihydropyridine binding to a dihydropyridine receptor protein. The arylazide-1,4-dihydropyridine[3H]azidopine binds with a Kd = 35 nM to a 50 kDa protein located in one of the internal cell membranes. Azidopine binding is fully reversible and can be partially inhibited by nimodipine and PN-200110. This protein is the first identified dihyropyridine receptor in an unicellular plant cell. It might serve as an internal calcium regulating channel in Chlamydomonas.

The management of cardiac arrhythmias: The role of encainide and sotalol panel discussion Session 3: Class III activity: The utility of sotalol

Dr. Brugada: We know that low potassium is associated with the side effects of sotalol and other antiarrhythmic agents as well. Potassium probably potentiates the effect on phase-zero conduction potential and also repolarization. Potassium alone prolongs repolarization and sotalol prolongs it further. However, I don't know if increasing potassium might have some additional effects. What is really important is the potassium gradient at the drug receptor site and that we cannot study.

A characterization of molecular similarity methods for property prediction

Molecular similarity methods are providing new tools for searching molecular databases, selecting compounds for drug screening, predicting molecular properties and modeling drug receptor sites (Johnson et al., 1987; Maggiora et al., 1987). This paper positions these methods amongst the more traditional a priori and algebraic methods for predicting property values and for finding a compound with a desired property value. Instances where these methods may complement each other are also noted.

Human cell mutants affected in the interaction of the 12β-OH group of cardiac glycosides with the digitalis receptor

The cross-resistance patterns of two different types of mutants of HeLa cells selected for resistance to the digoxin analog SC4453 (SC R mutants) in which the Na +/K +-ATPase is affected [A. Chopra and R.S. Gupta, J. biol. Chem. 261, 2034 (1986)], and towards numerous cardiac glycosides (CGs) and genins, were examined. One type of SC R mutant (designated as group C) was highly resistant to all CGs and genins investigated. In contrast, the other type of SC R mutant (group D) showed a high degree of cross-resistance towards selected CG derivatives (viz. digoxin, SC4453, digoxigenin, lanatoside C, α- and β-methyldigoxin, dihydrodigoxin, α- and β-acetyldigoxin, α,β-diacetyldigoxin), all of which contained a free 12β-OH group in the steroid structure. Slight cross-resistance of the group D mutants was also observed for other compounds (viz. ouabain, ouabagenin, dihydroouabain) that contain a free 11α-OH group in the molecule. However, these mutants exhibited no cross-resistance to other CG derivatives, which either lacked the above groups (viz. digitoxin, digitoxigenin, dihydrodigitoxin, digitoxigenin mono- and bisdigitoside, nerifolin, gitoxigenin, gitoxin. 16-acetylgitoxin, lanatosides A and B, cymarin, convallatoxin, oleandrin, strophanthidin, actodigin and bufalin) or in which the 12β- OH group was acetylated (viz. as in the case of 12-acetyldigoxin). Since the 12β-OH group is not required for CG-like activity, to account for these observations it is suggested that the genetic lesion in the group D mutant leads to the creation of a new binding site in the digitalis receptor, which specifically interacts with the 12β-OH group (the site presumably also interacts weakly with the 11α-OH group) and either prevents or distorts the binding of the compounds to the drug binding site on the receptor. Further investigations with the different classes of CG-resistant mutants at the molecular level should prove very useful in identifying the drug receptor site and in understanding how these drugs interact with it.

Distance geometry analysis of ligand binding to drug receptor sites

The method known as 'distance geometry approach' for receptor mapping procedures is discussed. In this method a ligand binding to a certain receptor is considered as a collection of ligand points. Binding sites of the receptor are either 'empty' or 'filled' site points; a ligand point might bind to an empty site point; filled site points indicate that at that point no binding is possible. A binding mode of a ligand is a list of which ligand points coincide with which empty binding sites. The applicability of the method for QSAR studies is discussed; as examples are mentioned the dihydrofolate reductase, beta 1- and beta 2-receptors. Finally, some ideas on future developments in receptor mapping are discussed.

Dual effects of dihydropyridines on whole cell and unitary calcium currents in single ventricular cells of guinea‐pig.

We studied the effects of dihydropyridine Ca channel ligands (DHPs), mainly nitrendipine and Bay K8644, on whole cell and single channel Ca currents on single myocytes isolated from the adult guinea-pig ventricle. Nitrendipine had dual effects, stimulatory or inhibitory, depending upon the membrane potential. At low frequencies (less than 0.03 Hz) and negative holding potentials (-90 mV or more), nitrendipine increased the Ca currents in a dose-dependent manner. The dose-response curve was best fitted by a Langmuir adsorption isotherm model which was the sum of two independent one-to-one drug-receptor sites with median effective doses (ED50S) of 1.0 X 10(-9) M and 1.4 X 10(-6) M respectively. When the membrane potential was held at -30 mV or less, nitrendipine inhibited the Ca currents, also in a dose-dependent manner. The dose-response curve was fitted by a single binding site model having a median inhibitor concentration (IC50) of 1.5 X 10(-9) M. At holding potentials between -70 and -40 mV, nitrendipine produced mixed effects on Ca currents; an increase occurred initially and this was followed by a decrease. When rundown was excluded, Bay K8644 showed only stimulatory effects on the Ca currents between holding potentials of -120 and -30 mV. When the test potential was zero or +10 mV the Ca currents reached peak values and the dose-response curve was best fitted by a single binding site model having an ED50 of 3 X 10(-8) M. When the effects were measured at negative test potentials of -30 to -10 mV, the curve was best fitted by a two-site model with ED50S of 3 X 10(-9) and 9 X 10(-7) M. At the single Ca channel level the stimulatory effect of nitrendipine was due to an increased probability that a Ca channel which had opened once would reopen, a reduction in records without activity and an increase in the mean open time. There were no changes in unit conductance. Inhibitory effects were due to a large increase in nulls. At lower concentrations the main effect of Bay K8644 was an increase in the probability of opening. At doses above 10(-6) M, a pronounced increase in the open time was observed. The effects we observed are attributed to at least two sites for DHP related to Ca channels; one with high affinity and one with a lower affinity. The low affinity site mediates a stimulatory effect due to greatly prolonged openings.(ABSTRACT TRUNCATED AT 400 WORDS)