Calcium-dependent inactivation of high voltage-activated Ca channels plays a crucial role in limiting rises in intracellular calcium (Ca). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming subunit. In contrast, little is known about how Ca can regulate low voltage-activated T-type Ca channels. Using whole cell patch clamp, we examined the biophysical properties of Ca current through the three T-type Ca channel isoforms, Ca3.1, Ca3.2, or Ca3.3, comparing internal solutions containing 27 nM and l μM free Ca Both activation and inactivation kinetics of Ca3.3 current in l μM Ca solution were more rapid than those in 27 nM Ca solution. In addition, both activation and steady-state inactivation curves of Ca3.3 were negatively shifted in the higher Ca solution. In contrast, the biophysical properties of Ca3.1 and Ca3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM (a calmodulin mutant that doesn't bind Ca) occluded the effects of l μM Ca on Ca3.3, implying that CaM is involved in the Ca regulation effects on Ca3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Ca3.3. Taken together, our results suggest that Ca3.3 T-type channel is potently regulated by Ca via interaction of Ca/CaM with the carboxyl terminus of Ca3.3.

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.

Ca2+ entry through Cav1.3 Ca2+ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Cav1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CTL) of rat Cav1.3L variant. Co-expression of Snapin with Cav1.3L/Cavβ3/α2δ2 subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Cav1.3S containing a short CT (CTS) in which a Snapin interaction site in the CTL was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Cav1.3L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Cav1.3L. Taken together, our results strongly suggest that Snapin directly interacts with the CTL of Cav1.3L, leading to up-regulation of Cav1.3L channel activity via facilitating channel opening probability.