Functional effects of splice variation on the neuronal alpha1D calcium channels.

摘要

Ca2+ enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Activation of inhibitory metabotropic receptors in pituitary GH₃ cells inhibits L-type channel (LTC) activity however the molecular identity of the channel remains unknown. Using RT-PCR, multiple alternatively spliced α _1D mRNAs were identified. Many of the α_1D-transcript variants encode “short” α_1D (α_1D-S ) subunits which have a QXXER amino acid sequence at their C-termini, a motif found in all other α₁ subunits that couple to opioid receptors. Subsequent sequencing of additional α_1D-S clones revealed an alternatively spliced transcript with a novel exon 9a (α _1D(+9a)). The resultant protein has a 29 amino acid insert within the intracellular loop linking domains I and II, near both the Ca 2+ channel β and G protein βγ subunit binding sites. The other splice variants identified terminate with a longer C-terminus that lacks the QXXER motif (α_1D-L). The predominant α _1D-S transcript variants in rat brain and GH₃ cells, their α _1D-L counterpart in GH₃ cells and the α_1D(+9a) isoform were cloned and expressed. All expressed α_1D clones lacked voltage-dependent G protein coupling yet exhibited voltage-dependent, G protein-independent facilitation when coexpressed with the β_2α subunit. The Ca2+ channel β subunit is known to affect the gating properties of the channel. An α₁-interaction domain with the β subunit has been identified on the intracellular loop between domains I and II of the α₁ subunit. Since exon 9a encodes for amino acids in this region, the gating properties of α _1D-subunit variants with (α_1D(+9a)) or without (α _1D(−9a)) this exon were investigated. The rates of activation and inactivation of Ba2+ currents increased according to the channel variant in the following order: α_1D(+9a)/β2α α_1D(−9a)/β2α α_1D(+9a) /β3 α_1D(−9a)/β3. Neither Ba 2+ nor Ca2+ currents mediated by α_1D(−9a) /β3 channels exhibited voltage-dependent facilitation. The inclusion of either the β2α subunit, or the exon 9a sequence in the α _1D subunit, or both was sufficient to reinstate facilitation. Thus α _1D subunit alternative splicing and β subunit gene switching alter Ca2+ channel gating properties in a manner that might influence activity-dependent changes in gene expression, a process which is highly regulated by influx of Ca2+ ions through LTC.