Ca²⁺ Regulation of Ca_v3.3 T-type Ca²⁺ Channel Is Mediated by Calmodulin

Abstract

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.

Calmodulin regulates Cav3 T-type channels at their gating brake

“…Consistent with our findings here, Lee et al ( 50 ) report a hyperpolarizing shift in activation and inactivation curves of Ca v 3.3 channels and faster inactivation kinetics, under conditions where intracellular free calcium is at a higher (1 μ m ) but not lower (27 n m ) calculated concentration. Lee et al ( 50 ) also observe a faster inactivation kinetics in Ca v 3.3 channels associated with external calcium compared with barium as the charge carrier.…”

“…Consistent with our findings here, Lee et al ( 50 ) report a hyperpolarizing shift in activation and inactivation curves of Ca v 3.3 channels and faster inactivation kinetics, under conditions where intracellular free calcium is at a higher (1 μ m ) but not lower (27 n m ) calculated concentration. Lee et al ( 50 ) also observe a faster inactivation kinetics in Ca v 3.3 channels associated with external calcium compared with barium as the charge carrier. The much faster inactivating mammalian T-type channels, Ca v 3.1 and Ca v 3.2, do not appear to exhibit the same calcium-dependent change in biophysical properties as Ca v 3.3 channels but possess a more pronounced voltage-dependent inactivation ( 61 ).…”

“…We illustrate using electron microscopy that purified full-length Ca v 3.1 binds calcium–CaM, data supported by co-immunoprecipitation experiments. A recent study by Lee et al reported that a rat Ca v 3.3 splice variant co-immunoprecipitates with CaM ( 50 ), adding support for a universality in CaM associating with all Ca v 3 T-type channels, in the same manner as CaM serves as a ubiquitous resident calcium sensor at the C-terminal IQ tails of Ca v 1 and Ca v 2 calcium channels ( 1 – 5 ) and Na v 1 sodium channels ( 6 ). Ca v 3 T-type channels (like all eukaryotic calcium and sodium channels), are large proteins (∼250 kDa) that do not readily express well enough for the purposes of extensive in vitro biochemical analyses.…”

“…The much faster inactivating mammalian T-type channels, Ca v 3.1 and Ca v 3.2, do not appear to exhibit the same calcium-dependent change in biophysical properties as Ca v 3.3 channels but possess a more pronounced voltage-dependent inactivation ( 61 ). Lee et al ( 50 ) illustrate that rat Ca v 3.3 channels possess a unique C-terminal sequence that associates with Ca 2+ –CaM, conferring the unique CaM regulation observed for Ca v 3.3 channels.…”

“…A suggested hypothesis is that all Ca v 3 channels possess a central, high-affinity Ca 2+ –CaM binding at the gating brake in the I–II linker, with additional modulatory, intracellular Ca 2+ –CaM-binding sites elsewhere, which can confer novel attributes, such as the Ca 2+ –CaM regulation at the C terminus for Ca v 3.3 channels ( 50 ). A highly customizable form of Ca 2+ –CaM regulation is observed within the “IQ motif centric” calcium (Ca v 1 and Ca v 2) and sodium (Na v 1) channels.…”