Single-cell RT-PCR and functional characterization of Ca²⁺ channels in motoneurons of the rat facial nucleus

Voltage-dependent Ca²⁺ channels are a major pathway for Ca²⁺ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltage-gated Ca²⁺ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca²⁺ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and ~5% of L-type. Despite the dominating role of P-type Ca²⁺ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca²⁺ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons. Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the α₁-subunit, we identified a highly heterogeneous expression pattern of Ca²⁺ channel α₁-subunit mRNA in individual neurons consistent with the Ca²⁺ current components found in the cell bodies and axon terminals. We detected mRNA for α(1A) in 86% of neurons, α(1B) in 59%, α(1C) in 18%, (α(1D) in 18%, and α(1E) in 59%. Either α(1A) or α(1B) mRNAs (or both) were present in all neurons, together with various other α₁-subunit mRNAs. The most frequently occurring combination was α(1A) with α(1B) and α(1E). Taken together, these results demonstrate that the Ca²⁺ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.