Functional properties of Cav1.3 (α1D) L-type Ca2+ channel splice variants expressed by rat brain and neuroendocrine GH3 cells

Ca2+ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca2+ entry through activation of pertussis toxin-sensitive G proteins leading to activation of K+ channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca2+ channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three alpha (1) (alpha (1A), alpha (1C), and alpha (1D)), four beta, and one alpha (2)-delta subunit in rat pituitary GH(3) cells; alpha (1B) and alpha (1S) transcripts were absent. GH(3) cells express multiple alternatively spliced alpha (1D) mRNAs. Many of the alpha (1D) transcript variants encode "short" alpha (1D) (alpha (1D-S)) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other al subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (alpha (1D-L)). We cloned and expressed the predominant alpha (1D-S) transcript variants in rat brain and GH(3) cells and their alpha (1D-L) counterpart in GH(3) cells. Unlike alpha (1A) channels, alpha (1D) channels exhibited current-voltage relationships similar to those of native GH(3) cell Ca2+ channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced alpha (1D) transcripts form functional Ca2+ channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of alpha (1D-S) subunit, is not sufficient to confer sensitivity to inhibitory G proteins.