Multisite phosphorylation of voltage-gated sodium channel alpha subunits from rat brain

Abstract

Reversible phosphorylation of ion channels underlies cellular plasticity in mammalian neurons. Voltage-gated sodium or Nav channels underlie action potential initiation and propagation, dendritic excitability, and many other aspects of neuronal excitability. Various protein kinases have been suggested to phosphorylate the primary or alpha subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav alpha subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in mediating diverse aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav alpha subunits affinity-purified from rat brain with two distinct monoclonal antibodies to identify 15 phosphorylation sites on Nav1.2, 12 of which have not been previously reported. We also found 3 novel phosphorylation sites on Nav1.1. In general, commonly used phosphorylation site prediction algorithms did not accurately predict these novel in vivo phosphorylation sites. Our results demonstrate that specific Nav alpha subunits isolated from rat brain are highly phosphorylated, and suggest extensive modulation of Nav channel activity in mammalian brain. Identification of phosphorylation sites using monoclonal antibody-based immunopurification and mass spectrometry is an effective approach to define the phosphorylation status of Nav channels and other important membrane proteins in mammalian brain.

Figure 1

Cartoon of the membrane topology of the Nav1.2 α subunit showing the phosphorylation sites identified in this study. Red dots mark the novel phosphorylation sites identified in this study. Green dots mark the phosphorylation sites identified here that had been identified in previous studies. Antibody symbols show general regions of K58/35 and K69/3 binding sites in the Nav1.2 α subunit.

Figure 2

A. Immuno-affinity purification of Nav α subunits. Coomassie brilliant blue-stained SDS gel of a large-scale immunopurification showing the yield of Nav α subunits obtained with mAbs K69/3 and K58/35. Left lane contains 500 ng per band of molecular weight standards; size noted to left of lane. Arrows denote positions of Nav α subunits and immunoglobulin heavy chain. B. Representative tandem mass spectrum of Nav1.2 peptide containing pS554. The spectrum of a doubly charged, singly phosphorylated Nav1.2 peptide at m/z 726.36 that was fragmented to produce a tandem mass spectrum with y-ion and b-ion series that described the sequence FSpSPHQSLLSIR (amino acids 552–563). The phosphorylation site was unambiguously assigned to S554 due to mass assignments from beta-eliminated b3, and y10²⁺ with neutral loss of phosphoric acid H₃PO₄ and y9, y9^2+, and y10²⁺ fragment ions.

Figure 3

In vivo phosphorylation sites and alternative peptides of rat brain Nav1.2 identified by nano-LC MS/MS analyses. A. Amino acid sequence of the canonical form of Nav1.2 (Swiss-Prot: P04775.1; NCBI Reference Sequence: NP_036779.1). Coverage obtained is highlighted in yellow, in vivo phosphorylation sites are in red, positions differing in an alternative Nav1.2 sequence (IPI00476429.2) are shown in blue. Underlined sequences represent the proposed transmembrane segments S1-S6 in each domain I-IV.

Figure 4

In vivo phosphorylation sites on rat brain Nav1.1 identified by nano-LC MS/MS analyses. A. Amino acid sequence of Nav1.1 (Swiss-Prot: P04774.1; NCBI Reference Sequence: NP_110502). Color scheme as in Figure 3.

Figure 5

Cartoon of the membrane topology of the Nav1.2 α subunit showing a comparison of in vivo phosphorylation sites versus predictions. The in vivo Nav1.2 phosphorylation sites described here are color coded to show their frequency of representation in the top 50 phosphorylation sites among four prediction algorithms (NetphosK, Netphos2, ScanSite and Disphos). Consensus phosphorylation sites where at least one of the protein kinase-specific prediction programs (NetPhosK, ScanSite) identified a specific protein kinase are given. Note that S579 (asterisk) is found only in the alternative IPI00476429.2 sequence.