Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis.
The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.
Lerner
Immanuel
I
Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Trus
Michael
M
Cohen
Roy
R
Yizhar
Ofer
O
Nussinovitch
Itzhak
I
Atlas
Daphne
D
D000313:Adrenal Medulla; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D007477:Ions / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000187:drug effects / Q000378:metabolism; D009865:Oocytes; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D011817:Rabbits; D051381:Rats; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D007477:Ions; D007811:Lanthanum; D002118:Calcium