Inhibition of voltage-gated calcium channels by sequestration of beta subunits.
The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.
Cuchillo-Ibañez
Inmaculada
I
Departamento de Farmacologi;a y Terapeutica, Facultad de Medicina, Universidad Autonoma de Madrid, 4, 28029, Arzobispo Morcillo, Spain.
Aldea
Marcos
M
Brocard
Jacques
J
Albillos
Almudena
A
Weiss
Norbert
N
Garcia
Antonio G
AG
De Waard
Michel
M
D000818:Animals; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism* / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology
D016827:CD8 Antigens; D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins