Ca2+ release by inositol 1,4,5-trisphosphate is blocked by the K(+)-channel blockers apamin and tetrapentylammonium ion, and a monoclonal antibody to a 63 kDa membrane protein: reversal of blockade by K+ ionophores nigericin and valinomycin and purification of the 63 kDa antibody-binding protein.
Ins(1,4,5)P3-induced Ca2+ release from platelet membrane vesicles was blocked by apamin, a selective inhibitor of low-conductance Ca(2+)-activated K+ channels, and by tetrapentylammonium ion, and was weakly inhibited by tetraethylammonium ion. Other K(+)-channel blockers, i.e. charybdotoxin, 4-aminopyridine and glybenclamide were ineffective. A monoclonal antibody (mAb 213-21) obtained by immunizing mice with the InsP3-sensitive membrane fraction from platelets also blocked Ca2+ release by InsP3 from membrane vesicles obtained from platelets, cerebellum, aortic smooth muscle, HEL cells and sea-urchin eggs. ATP-dependent Ca2+ uptake and binding of [3H]InsP3 to platelet membranes was unaffected by either K(+)-channel blockers or mAb 213-21. Blockade of Ca2+ release by apamin, tetrapentylammonium and mAb 213-21 was not affected by the Na+/H+ carrier monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), but could be completely reversed by the K+/H+ ionophore nigericin and partially reversed by the K+ carrier valinomycin. The antibody-binding protein (ABP) solubilized from platelets, cerebellum, and smooth muscle chromatographed identically on gel filtration, anion-exchange and heparin-TSK h.p.l.c. ABP was purified to apparent homogeneity from platelets and aortic smooth muscle as a 63 kDa protein by immunoaffinity chromatography on mAb 213-21-agarose. These results suggest that optimal Ca2+ release by InsP3 from platelet membrane vesicles may require the tandem function of a K+ channel. A counterflow of K+ ions could prevent the build-up of a membrane potential (inside negative) that would tend to oppose Ca2+ release. The 63 kDa protein may function to regulate K+ permeability that is coupled to the Ca2+ efflux via the InsP3 receptor.
Department of Pharmacology, University of Connecticut Health Center, Farmington 06032.
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O'Rourke
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Soons
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Flaumenhauft
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Watras
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Baio-Larue
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Matthews
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Feinstein
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000737:chemistry; D001011:Aorta / Q000737:chemistry; D001030:Apamin / Q000494:pharmacology*; D001792:Blood Platelets / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008970:Molecular Weight; D009550:Nigericin / Q000494:pharmacology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D013552:Swine; D014634:Valinomycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D008565:Membrane Proteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D000644:Quaternary Ammonium Compounds; D014634:Valinomycin; D001030:Apamin; D015544:Inositol 1,4,5-Trisphosphate; C027435:tetrapentylammonium; D009550:Nigericin; D002118:Calcium