Separate Binding and Functional Sites for ω co‐Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Abstract

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.

Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ channels

“…nM Wheeler et al, 1994 Owen et al, 1989;Rosario et al, 1989 . Indeed, while several groups including ours have consistently emphasized the lack of effect of v-conotoxin GVIA 2q Ž in depolarization-evoked Ca influx this paper and Ballesta et al, 1989;Owen et al, 1989;Rosario et al, . 1989;Duarte et al, 1993a , others-using single cell w 2q x Ca measurements-have reported the occurrence of i cell-specific and variable inhibitory effects of the toxin Ž .…”

“…observations . Furthermore, 1 mM is about 1000 times greater than the K for nitrendipine binding to bovine d Ž adrenomedullary membrane fragments Garcia et al, 1984;. Ballesta et al, 1989 .…”

The mechanism of calcium channel facilitation in bovine chromaffin cells.

“…As for the SVL action (Fig. IF) (Garcia et al 1984;Artalejo et al 1991), N-type (Ballesta et al 1989;Hans, Illes & Takeda, 1990;Artalejo, Perlman & Fox, 1992), P-type (Gandia, Albillos & Garcia, 1993a;Albillos, Garcia & Gandia, 1993;Artalejo et al 1994), and Q-type (Lopez et al 1994), contributing in different degrees to exocytosis.…”

Cardiac L‐type Ca2+ channel triggers transmitter release in PC 12 cells

“…In analogy to chromaffin cells, the N-channel in PC12 cells is not functional (shown in Fig, 1A). Chromaffin cells contain oCgTx binding sites [29,30] and w-CgTx-sensitive currents 131,321 but neither catecholamine secretion nor increase of [Ca2']i transients were antagonized by w-CgTx [33,34]. Fu~he~ore, synaptota~in, a small synaptic vesicleassociated protein which is involved in mediating transmitter release [35], is absolutely non-essential in the secretion process in PC12 cells [36].…”