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Biomedical subjects

V F King

Publications and source records attributed to V F King.

At least 19 recordsLinked to original sources
PubMed1992

Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.

Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1991

Characterization of high affinity binding sites for charybdotoxin in human T lymphocytes. Evidence for association with the voltage-gated K+ channel.

Charybdotoxin (ChTX) inhibits with high affinity a voltage-gated K+ channel that is present in human T lymphocytes. In this system, 125I-ChTX binds specifically and reversibly to a single class of sites which display a Kd of 8-14 pM, as measured by either equilibrium or kinetic binding protocols. The maximum density of sites, 542 sites/cell, correlates well with the density of K+ channel as determined by electrophysiological experiments. Binding of 125I-ChTX is modulated by the ionic strength of the incubation media and by Ca2+. Increasing concentrations of either K+, Na+, or Ca2+ cause inhibition of toxin binding. Inhibition of binding by Ca2+ is due, primarily, to an effect on toxin dissociation rates. Increasing the pH of the external media from 6.8 to 8.5 enhances toxin binding, due to an increase in affinity with no significant effect on the maximum density of receptor sites. Different agents that block the voltage-gated K+ channel in human T lymphocytes, inhibit toxin binding. Mitogen-stimulated T cells display 2.5-3-fold increase in toxin binding as compared with unstimulated control cells. These data, taken together, suggest that 125I-ChTX binding sites identified in this study, represent the predominant voltage-gated K+ channel present in peripheral human T lymphocytes. Therefore, 125I-ChTX is a useful probe for elucidating the physiological role of this type of K+ channel.

Binding Sites↗
PubMed1991

Use of toxins to study potassium channels.

Potassium channels comprise groups of diverse proteins which can be distinguished according to each member's biophysical properties. Some types of K+ channels are blocked with high affinity by specific peptidyl toxins. Three toxins, charybdotoxin, iberiotoxin, and noxiustoxin, which display a high degree of homology in their primary amino acid sequences, have been purified to homogeneity from scorpion venom. While charybdotoxin and noxiustoxin are known to inhibit more than one class of channel (i.e., several Ca(2+)-activated and voltage-dependent K+ channels), iberiotoxin appears to be a selective blocker of the high-conductance, Ca(2+)-activated K+ channel that is present in muscle and neuroendocrine tissue. A distinct class of small-conductance Ca(2+)-activated K+ channel is blocked by two other toxins, apamin and leiurotoxin-1, that share no sequence homology with each other. A family of homologous toxins, the dendrotoxins, have been purified from venom of various related species of snakes. These toxins inhibit several inactivating voltage-dependent K+ channels. Although molecular biology approaches have been employed to identify and characterize several species of voltage-gated K+ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K+ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest.

Amino Acid Sequence↗
PubMed1990

Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Charybdotoxin (ChTX), a potent peptidyl inhibitor of several types of K+ channels, binds to sites in vascular smooth muscle sarcolemma (Vázquez, J., Feigenbaum, P., Katz, G. M., King, V. F., Reuben, J. P., Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, M. L. (1989) J. Biol. Chem. 265, 20902-20909) which are functionally associated with a high conductance Ca2(+)-activated K+ channel (PK,Ca). 125I-ChTX also binds specifically and reversibly to a single class of sites in plasma membranes prepared from rat brain synaptosomes. These sites exhibit a Kd of 25-30 pM, as measured by either equilibrium or kinetic binding protocols and display a maximum density of about 0.3-0.5 pmol/mg of protein. Competition studies with native ChTX yield a Ki of 8 pM for the noniodinated toxin. The highest density of ChTX sites exists in vesicle fractions of plasma membrane origin. Binding of 125I-ChTX is modulated by metal ions that interact with K+ channels: Ba2+, Ca2+, and Cs+ cause inhibition of ChTX binding; Na+ and K+ stimulate binding at low concentration before producing complete inhibition as their concentration is increased. Stimulation of binding is due to an allosteric interaction that decreases Kd whereas inhibition results from an ionic strength effect. Tetraethylammonium ion has no effect on binding, but tetrabutylammonium ion blocks binding with a Ki of 2.5 mM. Different toxins (i.e. alpha-dendrotoxin, noxiustoxin) that inhibit an inactivating, voltage-dependent K+ channel (PK,V) block 125I-ChTX binding in brain. In marked contrast, iberiotoxin, a selective inhibitor of PK,Ca, has no effect on ChTX binding in this preparation. Inhibition of ChTX binding by alpha-dendrotoxin and noxiustoxin results from an allosteric interaction between separate binding sites for these agents and the ChTX receptor. Taken together, these results suggest that the ChTX sites present in brain are associated with PK,V rather than with PK,Ca. Therefore, 125I-ChTX is a useful probe for elucidating the biochemical properties of a number of different types of K+ channels.

Animals↗
PubMed1990

Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity.

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).

Amiloride↗
PubMed1989

Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel.

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.

Animals↗
PubMed1989

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex.

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.

Animals↗
PubMed1988

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 2. Mechanism of inhibition by bepridil.

Bepridil, an antiarrhythmic agent, inhibits Na-Ca exchange in cardiac sarcolemmal membrane vesicles (Ki = 30 microM) by a novel mechanism, different from that determined for amiloride analogues [Slaughter, R. S., Garcia, M. L., Cragoe, E. J., Jr., Reeves, J. P., & Karczorowski, G. J. (1988) Biochemistry (preceding paper in this issue)]. Bepridil causes partial inhibition of Nai-dependent Ca2+ uptake but complete block of Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange is noncompetitive vs Ca2+ but competitive vs Na+ in both K+ and sucrose. Bepridil also blocks Ca-Ca exchange, with or without K+ present. However, K+ has two effects on inhibition: it reduces the potency of bepridil and causes inhibition to become partial. Inhibition of Ca-Ca exchange displays noncompetitive kinetics vs Ca2+ in either sucrose or K+. Dixon analyses of Na-Ca exchange inhibition caused by mixtures of bepridil and amiloride analogues demonstrate that these compounds produce a competitive interaction at a common carrier site that is evident only at low concentrations of amiloride inhibitors. Hill plots of bepridil inhibition of Na-Ca and Ca-Ca exchange display unitary Hill coefficients. These results indicate that bepridil interacts at only one substrate-binding site, the site selective for Na+, where amiloride analogues also preferentially interact. However, unlike amiloride, bepridil does not interact at the common Na+, Ca2+-binding site of the carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Interaction of tetrandrine with slowly inactivating calcium channels. Characterization of calcium channel modulation by an alkaloid of Chinese medicinal herb origin.

Tetrandrine, a bis-benzylisoquinoline alkaloid derived from the Chinese medicinal herb Stephania tetrandra, is a putative Ca2+ entry blocker whose mechanism of action is unknown. To investigate this mechanism, the effects of tetrandrine were characterized on binding of three chemical classes of Ca2+ entry blockers in cardiac sarcolemmal membrane vesicles. In the range 25-37 degrees C, tetrandrine completely blocks diltiazem binding, partially inhibits D-600 binding, and markedly stimulates nitrendipine binding, with greatest enhancement occurring at 37 degrees C. The potency of tetrandrine is increased 10-fold as temperature is raised from 25 to 37 degrees C. Scatchard analyses indicate that inhibition of diltiazem binding and stimulation of nitrendipine binding result from changes in ligand affinities while inhibition of D-600 binding is due to both an increase in KD and decrease in Bmax of aralkylamine receptors. Ligand dissociation studies reveal that tetrandrine increases D-600 off-rates, decreases nitrendipine off-rates, but has no effect on diltiazem dissociation kinetics. In addition, tetrandrine reversibly blocks inward Ca2+ currents through L-type Ca2+ channels in GH3 anterior pituitary cells. These results indicate that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+ entry blocker receptor complex and allosterically modulates ligand binding at other receptors in this complex. These findings suggest that tetrandrine is a structurally unique natural product Ca2+ entry blocker and provide a rationale explanation for the therapeutic effectiveness of this agent.

Alkaloids↗
PubMed1988

Interactions of DPI 201-106, a novel cardiotonic agent, with cardiac calcium channels.

The interaction of DPI 201-106, a novel cardiotonic agent, with the calcium entry blocker receptor complex was studied using porcine cardiac sarcolemmal membranes. DPI 201-106 and the chemically-related calcium antagonist, cinnarizine, produce concentration-dependent inhibition of nitrendipine, gallopamil and diltiazem binding to their respective sites in these vesicles. This effect of DPI 201-106 is not stereoselective since resolved stereoisomers of this compound display equal potency in inhibiting each of the binding reactions. Equilibrium ligand binding studies revealed that DPI 201-106 and cinnarizine cause mixed inhibitory patterns at the aralkylamine and benzothiazepine sites (i.e. both Kd and Bmax values were affected) while mainly increasing Kd at the dihydropyridine site. The kinetics of ligand dissociation from the three calcium entry blocker receptors, together with measurements of dihydropyridine association kinetics, further demonstrate that DPI 201-106 interacts at a unique site in the receptor complex and allosterically modulates binding of nitrendipine, gallopamil and diltiazem. The functional consequences of the above interactions with the calcium channel were studied in isolated cardiac preparations. In guinea-pig atria, DPI 201-106 increased force of contraction. This inotropic effect is seen only with the S(-) enantiomer and is unaltered by nitrendipine-, verapamil- or diltiazem-pretreatment, indicating DPI 201-106 does not act as a stimulant of this channel. Furthermore, DPI 201-106 did not alter the inotropic action of Bay K 8644, a calcium channel stimulant. Spontaneous rate of guinea-pig right atria is decreased by both DPI 201-106 and cinnarizine. In addition, potassium-induced contractures in cat papillary muscles are reduced by both agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

L-652,469--a dual receptor antagonist of platelet activating factor and dihydropyridines from Tussilago farfara L.

L-652,469, 14-acetoxy-7 beta-(3'-ethylcrotonoyloxy)-notonipetranone, isolated from the methylene chloride extracts of the buds of Tussilago farfara L, was found to inhibit both platelet activating factor (PAF) and Ca2+ entry blocker binding to membrane vesicles. It inhibits the [3H]PAF specific binding to rabbit platelet membranes with equilibrium inhibition constants (Ki) of 3.2 and 4.0 microM in the presence of 150 mM NaCl and 10 mM MgCl2 respectively. It is a competitive PAF receptor antagonist with an equilibrium dissociation constant (KB) of 5.16 microM. It also competitively inhibits the specific binding of Ca2+ channel blockers (e.g. [3H]nitrendipine; Ki = 1.2 microM) in cardiac sarcolemmal vesicles. At 10(-5) M, L-652,469 causes a 60% relaxation of Ca2+-induced contraction of rat thoracic aorta strips. Due to its dual antagonistic activities, L-652,469 potently inhibits the gel-filtered rabbit platelet aggregation with a pA2 of 5.79. It was also found to be orally active with a beneficial effect to inhibit the PAF-induced rat foot edema and the first phase of carrageenan-induced rat hindpaw edema.

Animals↗
PubMed1986

Binding of Ca2+ entry blockers to cardiac sarcolemmal membrane vesicles. Characterization of diltiazem-binding sites and their interaction with dihydropyridine and aralkylamine receptors.

Stereospecific saturable and reversible binding of d-cis-diltiazem has been demonstrated in cardiac sarcolemmal membrane vesicles. Analysis of binding by either equilibrium or kinetic techniques indicates the presence of a single class of benzothiazepine receptors which bind diltiazem with a KD of 80 nM at 25 degrees C. Benzothiazepine receptors copurify with other sarcolemmal marker activities and exist in a complex with distinct receptors for dihydropyridine and aralkylamine Ca2+ entry blockers in a 1:1:1 stoichiometry. Ligand binding to one receptor of this complex influences binding reactions at the other two sites in a manner that depends on ambient temperature. Binding of either dihydropyridine agonists or antagonists causes partial inhibition of diltiazem binding at 25 degrees C (Bmax effect), while most dihydropyridine antagonists stimulate and agonists inhibit diltiazem binding at 37 degrees C (both are KD effects). This temperature-dependent change in receptor coupling was confirmed by Scatchard analyses and study of diltiazem dissociation kinetics. Verapamil, interacting at the aralkylamine receptor, inhibits diltiazem binding equivalently at 25 and 37 degrees C (KD effects). In addition, both classes of dihydropyridines inhibit verapamil binding in a temperature-independent fashion, as does diltiazem (all are KD effects). Allosteric coupling between benzothiazepine and dihydropyridine receptors is manifested in cardiac muscle since the negative inotropic potency of diltiazem is increased by nitrendipine and decreased by 4-(O-trifluromethy(phenyl)-2,6-dimethyl-5-nitro-1,4-dihydropyridin e-3- carboxylic acid, methyl ester. These results suggest a model in which the Ca2+ entry blocker receptor complex undergoes a change between 25 and 37 degrees C so that at the latter temperature all sites are directly coupled. Allosteric coupling may have important consequences in vivo since it can be detected in functional assays of Ca2+ channel activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

Differential binding of folates by rat renal cortex brush border and basolateral membrane preparations.

The binding of radioactive 5-methyltetrahydrofolate and folic acid was found to be greater in brush border than in basolateral membrane preparations of rat renal cortex. This appeared to be due to an increased amount of a specific folate binding protein in the brush border membrane preparations as compared to those of the basolateral membrane. The binding was saturable and inhibited by nonradioactive folic acid and, therefore, a specific, rather than nonspecific process. The Km's for folic acid binding in brush border and basolateral membrane preparations were similar and involved a single high-affinity binding site. In contrast, methotrexate was found to bind equally well to both brush border and basolateral membrane preparations. Moreover, folic acid binding was not inhibited by an equimolar amount of methotrexate. A folate binding protein could be extracted from either membrane preparation with 1% Triton X-100 and, to a lesser extent, with 0.6 M NaCl. These different extraction procedures resulted in different apparent molecular weights for folate binding protein (greater than 160,000 for Triton X-100-extracted samples and 40,000 for NaCl-extracted samples). The membrane preparation pellets remaining after NaCl extraction were able to rebind tritiated folic acid and also the 40,000-Da folate binding protein. On the other hand, membrane preparations extracted with Triton X-100 lost the ability to bind folic acid or the 40,000-Da folate binding protein. These differences in molecular weight and rebinding capacity may be explained by the existence of a receptor for folate binding protein which was extracted by Triton X-100, but not by NaCl. The greater concentration of folate binding protein in the renal tubule cell brush border membrane preparations as compared to those from basolateral membranes ascribes, for the first time, a functional role for folate binding protein in the renal reabsorption of folates which is required to prevent loss of folate in the urine and perhaps in the membrane transport of folates in general.

Animals↗
PubMed1982

Urate uptake in membrane vesicles of rat renal cortex: effect of copper.

[2-14C]Urate uptake was studied in brush border and basolateral membrane vesicles of rat renal cortex. In the absence of copper, urate equilibrated without metabolism of transported urate. Exposure of the vesicles to copper significantly stimulated uptake, and in these vesicles uptake was also stimulated by NaCl or KCl gradients. Allantoin accumulated in these vesicles due to oxidation of transported urate. This oxidation is ascribed to a copper-stimulated, membrane-associated uricase since purified uricase and the membranes had similar Km values, both were inhibited by oxonic acid, and extramembranal uricase was not detected. Oxonic acid and pyrazinoic acid inhibited both uptake and enzyme activity. These findings suggest that urate uptake is carrier mediated and that uricase may play some role in transport. In addition, it appears that a significant loss of copper occurs during isolation of membrane vesicles that profoundly affects the characteristics of urate uptake. Those properties of the membrane that influence urate uptake, however, can be restored by exposure of the membranes to copper.

Absorption↗