The Cav3.2 T-type calcium channel is implicated in various pathological conditions, including cardiac hypertrophy, epilepsy, autism, and chronic pain. Phosphorylation of Cav3.2 by multiple kinases plays a pivotal role in regulating its calcium channel function. The calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, interacts physically with Cav3.2 and modulates its activity. However, it remains unclear whether calcineurin dephosphorylates Cav3.2, the specific spatial regions on Cav3.2 involved, and the extent of the quantitative impact. In this study, we elucidated the serine/threonine residues on Cav3.2 targeted by calcineurin using quantitative mass spectrometry. We identified six serine residues in the N-terminus, II-III loop, and C-terminus of Cav3.2 that were dephosphorylated by calcineurin. Notably, a higher level of dephosphorylation was observed in the Cav3.2 C-terminus, where calcineurin binds to this channel. Additionally, a previously known CaMKII-phosphorylated site, S1198, was found to be dephosphorylated by calcineurin. Furthermore, we also discovered that a novel CaMKII-phosphorylated site, S2137, underwent dephosphorylation by calcineurin. In CAD cells, a mouse central nervous system cell line, membrane depolarization led to an increase in the phosphorylation of endogenous Cav3.2 at S2137. Mutation of S2137 affected the calcium channel function of Cav3.2. Our findings advance the understanding of Cav3.2 regulation not only through kinase phosphorylation but also via calcineurin phosphatase dephosphorylation.

Background: We investigated ion channels at the skin, including peripheral nerve endings, which serve as output machines and molecular integrators of many pruritic inputs mainly received by multiple G protein-coupled receptors (GPCRs). Methods: Based on the level of chronic kidney disease–associated pruritus (CKD-aP), subjects were divided into two groups: non-CKD-aP (no or slight pruritus; n=12) and CKD-aP (mild, moderate, or severe pruritus; n=11). Skin samples were obtained from the forearm or elbow during operations on arteriovenous fistulas. We measured ion channels expressed at the skin, including peripheral nerve endings by RT-PCR: Nav1.8, Kv1.4, Cav2.2, Cav3.2, BKCa, Anoctamin1, TRPV1, TRPA1, and ASIC. Results: Expression of Cav3.2, BKCa, and anoctamin1 was significantly elevated in patients with CKD-aP. On the other hand, expression of TRPV1 was significantly reduced in these patients. We observed no significant difference in the levels of Cav2.2 or ASIC between subjects with and without CKD-aP. TRPA1, Nav1.8，and Kv1.4 were not expressed. Conclusions: It was concluded that this greater difference in expression of ion channels at the skin tissue including specific for cutaneous peripheral nerve endings in CKD patients with CKD-aP may increase generator potential related to itching.