Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit.
The contribution of voltage-gated calcium channels to excitable cell function depends, critically, upon the mechanisms that control their expression at the cell surface. While co-assembly of the pore forming alpha(1) and auxiliary beta subunits enhances channel surface expression, the levels are still only 30-40% of those seen with the core alpha(1B)/beta(1b)/alpha(2)delta calcium channel complex. To rationalize this observation, it has been suggested that the alpha(2)/delta subunit might stabilize calcium channel expression at the cell surface. To test this notion, we have resolved the effect of the alpha(2)/delta subunit on the rates of binding, internalization and degradation of defined N-type calcium channel surface complexes expressed in HEK293 cells, through pulse-labeling with the selective, cell impermeable, radioligand [(125)I]-omega-CgTx. Through detailed kinetic and sensitivity analysis we show that alpha(1B)/beta(1b)/alpha(2)delta complexes are internalized slowly (k(int) 0.4/h), whereupon, most become degraded (k(deg) 0.02/h). In contrast, alpha(1B)/beta(1b) complexes are internalized more rapidly (k(int) 0.8/h), following which they are either quickly degraded (k(deg) 0.1/h) or are sequestered slowly (k(tra) 0.1/h) to a pool that is metabolically stable within the time-frame of our experiments (24h). In neither case did we find evidence for recycling via the cell surface. Thus, our data argue for a novel mechanism where complexes lacking an alpha(2)/delta subunit are cleared from the cell surface and are rapidly degraded or stored, possibly for further attempts at complexation as new alpha(2)/delta subunits become available. The slower rate of internalization of complexes containing the alpha(2)/delta subunit rationalizes the stabilizing effect this subunit has upon calcium channel surface expression and suggests a mechanism by which alpha(2)delta mutations may cause severe neurological deficits.
Bernstein
Geula M
GM
Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network. 399 Bathurst Street, Toronto, Ontario, Canada
Jones
Owen T
OT
D000818:Animals; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D021122:Protein Subunits; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020866:omega-Conotoxin GVIA / Q000378:metabolism
C486060:Cacna2d1 protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D020866:omega-Conotoxin GVIA