Temperature-dependent functional inversion of Ca_v3.2 channels during repetitive activity

Temperature strongly influences neuronal excitability and voltage-gated ion channel function. Ca_v3.2 T-type calcium channels are highly expressed in dorsal root ganglion and trigeminal ganglion neurons, where they contribute to nociceptive signaling, cold hypersensitivity, and neuropathic pain. However, how temperature influences Ca_v3.2 function during repetitive neuronal activity remains incompletely understood. Here, we investigated the effects of temperature on Ca_v3.2 gating and calcium influx using whole-cell voltage-clamp recordings performed at 37 °C, 22 °C, and 13 °C. Cooling markedly reduced macroscopic current amplitude under conventional voltage-step protocols while profoundly slowing activation, inactivation, deactivation, and recovery from inactivation kinetics. In contrast, steady-state voltage dependence was comparatively less affected, revealing highly non-uniform temperature sensitivity across distinct gating transitions. During trains of action-potential-like waveforms delivered at 10-100 Hz, increasing stimulation frequency progressively inverted the temperature dependence of Ca_v3.2-mediated calcium influx. Although cooling reduced peak current amplitude, it promoted sustained channel activity and enhanced cumulative calcium influx during high-frequency stimulation. This functional inversion resulted from disproportionate slowing of channel gating kinetics, particularly inactivation and deactivation. A kinetically slowed Ca_v3.2 variant (V416R) reproduced key low-temperature kinetic features and markedly attenuated this inversion. Together, these findings identify a previously unrecognized temperature-dependent functional inversion of Ca_v3.2 channels during repetitive activity with potentially important pathophysiological implications.