Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit

Abstract

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.