title: Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.
abstract: P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca(V)2.1 alpha1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca(V)2 channels and suggest that the ability of individual Ca2+ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.
authors.affiliation: Department of Biomedical Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Robert C E
authors.initials: RC
authors.lastname: Wykes
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Bauer
authors.forename: Saeed U
authors.initials: SU
authors.lastname: Khan
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Weiss
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: Seward
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020865:Calcium Channels, P-Type / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D025802:Electric Capacitance; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D020864:Calcium Channels, N-Type; D020865:Calcium Channels, P-Type; D002147:Calmodulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium