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

Ca v 3.1 T‐type Ca 2+ 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 Ca v 3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca v 3.1 channel has been poorly investigated. In this work, we analyzed rat Ca v 3.1 protein expressed in HEK‐293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca v 3.1 phosphorylation map which includes the reported mouse Ca v 3.1 phosphosites. Site‐directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho‐silent Ca v 3.1 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 Ca v 3.1 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 Ca v 3.1 at the N‐ and C‐terminal regions play crucial roles in the regulation of the channel activity and voltage‐dependent gating properties.