The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Cav1.2 (Lc-type) and Cav2.3 (R-type)

Roy Cohen, Lisa Elferink, Daphne Atlas

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a C2+ sensor for neuronal and neuro-endocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neuro-transmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 α1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.

Original languageEnglish (US)
Pages (from-to)9258-9266
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number11
DOIs
StatePublished - Mar 14 2003

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Synaptotagmins
Polylysine
Kinetics
Syntaxin 1
Electric potential
Calcium
Mutation
Transmitters
Synaptotagmin I
Neurosecretion
Qa-SNARE Proteins
PC12 Cells
Secretory Vesicles
Synaptic Transmission
Neurotransmitter Agents
Molecular Biology
Chemical activation
Sensors

ASJC Scopus subject areas

  • Biochemistry

Cite this

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Cav1.2 (Lc-type) and Cav2.3 (R-type). / Cohen, Roy; Elferink, Lisa; Atlas, Daphne.

In: Journal of Biological Chemistry, Vol. 278, No. 11, 14.03.2003, p. 9258-9266.

Research output: Contribution to journalArticle

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abstract = "Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a C2+ sensor for neuronal and neuro-endocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neuro-transmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 α1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.",
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