Identification of T-type α1H Ca²⁺ channels (Ca_v3.2) in Major pelvic ganglion neurons

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca²⁺ channels. To date, the T-type Ca²⁺ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca²⁺ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca²⁺ as a charge carrier, T-type Ca²⁺ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca²⁺ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca²⁺ with 10 mM Ba²⁺ did not affect the amplitudes of T-type Ca²⁺ currents. Mibefradil, a known T-type Ca²⁺ channel antagonist, depressed T-type Ca²⁺ currents in a concentration-dependent manner (IC₅₀ = 3 μM). Application of Ni²⁺ also produced a concentration-dependent blockade of T-type Ca²⁺ currents with an IC₅₀ of 10 μM. The high sensitivity to Ni²⁺ implicates α1H in generating the T-type Ca²⁺ currents in MPG neurons. RTPCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of α1H (called pelvic Ta and Tb, short and long forms of α1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca²⁺ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 μM Ni²⁺ that blocked 50% of T-type Ca²⁺ currents and had a little effect on HVA Ca²⁺ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 μM Ni²⁺ that blocked most of the T-type Ca²⁺ currents. In conclusions, T-type Ca²⁺ currents in MPG neurons mainly arise from α1H among the three isoforms (α1G, α1H, and α1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.