Mass-spec analysis reveals phosphorylation sites in Na_v1.2

Berendt and co-workers published a manuscript in the Journal of Proteome Research that looked at phosphorylation in brain Na_v1.x channels using affinity purification, tryptic digestion, and mass spec of the generated peptides (J Proteome Res, EPub 23Feb2010, PMID: 20131913). The antigenicity of the antibodies was such that one was highly selective for Na_v1.2 (96 % selective vs other Na_v's), while the second antibody detected all Na_v channels. Fifteen reversible phosphorylation sites were identified in peptides unique to Na_v1.2; three were found in Na_v1.1 fragments, all of which analogous to sites in Na_v1.2. Some sites identified in Na_v1.2 may have been missed, as the peptide coverage did not entirely overlap with all candidate sites in Na_v1.1 as determined by sequence alignment with Na_v1.2 (specially marked below).

Earlier biochemical work assessing Na_v phosphorylation

The Berendt et al. study confirms sites identified in earlier biochemical work by the Catterall group (Murphy & Catterall, JBC 267:16129, 1992 and Murphy et al., JBC 268:27355, 1993) looking at protein kinase C and A, respectively. A more recent analysis also identified several Fyn kinase sites in Na_v1.2 (Beacham et al, J Neurosci 27:11543, 2007). There is furthermore a report by Brechet et al. (J Cell Biol 183:1101, 2008) that suggests phosphorylation of three sites via casein kinase II.

Na_v1.1 phosphorylation sites not associated with epilepsy

To date, none of residues of interest has been associated with epilepsy. None of the phosphorylation sites lies within the Na_v1.1 variable splicing region.

Phosphorylation sites in Na_v1.1

The following list identifies the phosphorylation sites in Na_v1.1 (GenBank number P35498). Only sites that were either directly confirmed in the Berendt study or candidate sites indicated either by comparison to Na_v1.2 in the same publication or in analogy to older studies as indicated but not covered the Berendt study are included. Three-letter amino acid code followed by residue position with the starting methionine = 1.

• Ser470
• Ser480*
• Ser525*
  
• Ser551
• Ser570* (deduced from JBC 267:16129 and JBC 286:27355)
• Ser607
• Ser1122* (deduced from JCB 183:1101, 2008)
• Ser1516* (deduced from JBC 267:16129)

Residues in bold are confirmed in vivo phosphorylation sites by the Berendt study. A single asterisk indicates possible phosphorylation based on paralog data, and lack of peptide coverage in the Berendt study, so the question remains whether or not these are phosphorylated in Na_v1.1 or not.