Functional properties of Cav1.3 (alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and neuroendocrine GH3 cells.
Ca(2+) enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca(2+) 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 Ca(2+) 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 alpha(1) 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(lD-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 Ca(2+) channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced alpha(1D) transcripts form functional Ca(2+) 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.
Safa
P
P
Department of Pharmacology, The George Washington University, Washington, DC 20037, USA.
Boulter
J
J
Hales
T G
TG
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D020867:omega-Agatoxin IVA / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D020033:Protein Isoforms; D012333:RNA, Messenger; D011957:Receptors, Opioid; D011994:Recombinant Proteins; D020867:omega-Agatoxin IVA; D011188:Potassium