Phosphorylation of the Cav3.2 T-type calcium channel directly regulates its gating properties.
Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France; LabEx Ion Channel Science and Therapeutics, Montpellier F-34094, France;
Iulia
I
Blesneac
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France; LabEx Ion Channel Science and Therapeutics, Montpellier F-34094, France;
Jean
J
Chemin
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France; LabEx Ion Channel Science and Therapeutics, Montpellier F-34094, France;
Isabelle
I
Bidaud
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France; Laboratoire Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, CNRS UMR 5235, Montpellier F-34095, France.
Sylvaine
S
Huc-Brandt
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France;
Franck
F
Vandermoere
Université de Montpellier, CNRS UMR 5203, Département de Neuroscience & Biologie des Canaux Ioniques, Institut de Génomique Fonctionnelle, Montpellier F-34094, France; INSERM, Montpellier F-34094, France; LabEx Ion Channel Science and Therapeutics, Montpellier F-34094, France; philippe.lory@igf.cnrs.fr.
Philippe
P
Lory
D020747:Calcium Channels, T-Type / Q000378:metabolism* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D018408:Patch-Clamp Techniques; D010766:Phosphorylation
C479952:CACNA1H protein, human; D020747:Calcium Channels, T-Type
Cav3.2 subunit; T-type calcium channel; mass spectrometry; patch clamp; phosphorylation