Separate binding and functional sites for omega-conotoxin and nitrendipine suggest two types of calcium channels in bovine chromaffin cells.
Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.
Ballesta
J J
JJ
Departamento de Neuroquímica, Universidad de Alicante, Spain.
Palmero
M
M
Hidalgo
M J
MJ
Gutierrez
L M
LM
Reig
J A
JA
Viniegra
S
S
Garcia
A G
AG
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008978:Mollusk Venoms / Q000378:metabolism* / Q000494:pharmacology; D009568:Nitrendipine / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D008978:Mollusk Venoms; D011978:Receptors, Nicotinic; D020960:omega-Conotoxins; C053423:Conus magus toxin; D009568:Nitrendipine; D002118:Calcium