Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T‐type Ca2+ channels

Cav3.1 T‐type Ca2+ channels play pivotal roles in neuronal low‐threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Cav3.1 channels. However, systematic identification of phosphorylation sites (phosphosi...

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Bibliographic Details
Published in:Journal of cellular physiology 2023-01, Vol.238 (1), p.210-226
Main Authors: Jeong, Sua, Shim, Ji Seon, Sin, Seok Kyo, Park, Kang‐Sik, Lee, Jung‐Ha
Format: Article
Language:English
Subjects:
Amplitudes
Calcium channels
Calcium channels (voltage-gated)
Calcium ions
Cav3.1 T‐type Ca2+ channel
Channel gating
Channels
Electric potential
Firing pattern
Functional analysis
Gametocytes
Kinases
Kinetics
Mass spectrometry
Mass spectroscopy
Mutation
Oocytes
Pacemakers
Phosphorylation
Residues
Site-directed mutagenesis
surface expression
Voltage
voltage‐clamping
Xenopus oocytes
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Summary:Cav3.1 T‐type Ca2+ channels play pivotal roles in neuronal low‐threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Cav3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Cav3.1 channel has been poorly investigated. In this work, we analyzed rat Cav3.1 protein expressed in HEK‐293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Cav3.1 phosphorylation map which includes the reported mouse Cav3.1 phosphosites. Site‐directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho‐silent Cav3.1 mutants expressed in Xenopus oocytes showed that the phospho‐silent mutation of the N‐terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho‐silent mutations of the C‐terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage‐dependent gating properties. In contrast, the phosphomimetic Asp mutations of Cav3.1 on the N‐ and C‐terminal Ser residues reversed the effects of the phospho‐silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Cav3.1 at the N‐ and C‐terminal regions play crucial roles in the regulation of the channel activity and voltage‐dependent gating properties.
ISSN:0021-9541
1097-4652
DOI:10.1002/jcp.30920