title: The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation.
abstract: Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cac(ts2) mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cac(ts2) mutation occurs in the cytoplasmic carboxyl tail of the alpha1-subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+-imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cac(ts2) mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Ca(v)2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 degrees C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Ca(v)2.1 channel inactivation.
authors.affiliation: Department of Physiology, University of Toronto, Toronto, Ontario, Canada. gmacleod@neurobio.arizona.edu
authors.forename: G T
authors.initials: GT
authors.lastname: Macleod
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Karunanithi
authors.forename: J B
authors.initials: JB
authors.lastname: Peloquin
authors.forename: H L
authors.initials: HL
authors.lastname: Atwood
authors.forename: J E
authors.initials: JE
authors.lastname: McRory
authors.forename: G W
authors.initials: GW
authors.lastname: Zamponi
authors.forename: M P
authors.initials: MP
authors.lastname: Charlton
mesh_terms: D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001522:Behavior, Animal / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007814:Larva / Q000033:anatomy & histology / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D009469:Neuromuscular Junction / Q000502:physiology; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D009435:Synaptic Transmission / Q000502:physiology; D013696:Temperature
chemical_list: D000814:Aniline Compounds; D002118:Calcium; D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D029721:Drosophila Proteins; D005452:Fluoresceins; D021122:Protein Subunits; C503942:Oregon green 488 BAPTA-1; C472405:cac protein, Drosophila