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NEUROSCIENCE |
1 Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, University College London, London, UK
2 School of Crystallography, Birkbeck College, London, UK
The CaVß subunits of voltage-gated calcium channels regulate the trafficking and biophysical properties of these channels. We have taken advantage of mutations in the tyrosine residue within the alpha interaction domain (AID) in the I–II linker of CaV2.2 which reduce, but do not abolish, the binding of ß1b to the AID of CaV2.2. We have found that the mutation Y388S decreased the affinity of CaVß1b binding to the CaV2.2 I–II linker from 14 to 329 nM. However, the Y388S mutation had no effect on current density and cell surface expression of CaV2.2/
2
-2/ß1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of CaVß1b for the Y388S I–II linker of CaV2.2, all the key features of modulation as well as trafficking by CaVß subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the CaV2.2 I–II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when CaVß1b cDNA was diluted 50-fold, all evidence of interaction with CaV2.2 Y388S was lost, although wild-type CaV2.2 was still normally modulated by the reduced concentration of ß1b. These results indicate that high affinity interaction with the 1 subunit is not necessary for any of the modulatory effects of CaVß subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the CaVß subunit is present in sufficient excess.
(Received 15 March 2006;
accepted after revision 13 April 2006;
first published online 20 April 2006)
Corresponding author A. C. Dolphin: Laboratory of Cellular and Molecular Neuroscience, Department of Pharmacology, Andrew Huxley Building, University College London, Gower Street, London, WC1E 6BT, UK. Email: a.dolphin{at}ucl.ac.uk
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