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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 α subunit of Nav channels, affecting diverse aspects of channel function. Previous studies of Nav α subunit phosphorylation have led to the identification of a small set of phosphorylation sites important in meditating aspects of Nav channel function. Here we use nanoflow liquid chromatography tandem mass spectrometry (nano-LC MS/MS) on Nav α 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 α 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 important membrane proteins in mammalian brain.
The electrical excitability of cells is mediated by the expression of a large number of different types of plasma membrane ion channels. In mammalian neurons the distinct patterns of expression, localization and activity of different ion channels generates a wide array of firing patterns, determining the input-output relationship of neurons and their subcellular compartments. As such, dynamic modulation of ion channels by reversible phosphorylation underlies plasticity in neuronal signaling. Among ion channels, voltage-gated sodium or Nav channels generate a depolarizing inward current and determine the threshold for axonal action potential generation1, as well as the excitability of specific dendritic branches2, and aspects of synaptic integration3. Nav channels consist of a highly posttranslationally modified α subunit of approximately 2000 amino acids (Mr≈260 kDa), comprising four internally repeated homologous domains or pseudosubunits (I-IV), each of which contains six transmembrane segments (S1–S6) that act as the voltage-sensing and pore-forming domains 1. The short intracellular linker connecting pseudosubunits III and IV (i.e., the interdomain or ID III-IV linker) serves as the inactivation gate, folding into the channel structure and blocking the pore from the inside during sustained depolarization of the membrane 1. Nav α subunits are associated with one or two smaller auxiliary β subunits with a molecular weight of 30–40 kDa 4. Although the α subunit is sufficient for functional expression 5, β subunits modify the kinetics and voltage dependence of channel gating 4.
Ten genes located on four chromosomes encode the different subtypes of Nav channel α subunits1. At least nine of these genes are expressed in the nervous system6, with the exception of Nav1.4, which is expressed predominantly in adult skeletal muscle7. Of the different channel subtypes, Nav1.1, Nav1.2, Nav1.3 and Nav1.6 are prominently expressed in the mammalian brain, with different spatial and temporal patterns of expression 6. Among these Nav1.2 is the most abundant α subunit expressed in the central nervous system, comprising approximately 80% of the total rat brain Nav α subunit pool 8. Based on its prominent localization in axons and nerve terminals 9, 10, Nav1.2 is expected to control conductance of axonal action potentials and neurotransmitter release in presynaptic terminals 11. In contrast, Nav1.1 is primarily localized in the soma and proximal dendrites9, 10, where it plays a role in neuronal excitability through integration of synaptic impulses to set the threshold for action potential initiation and propagation in dendritic and axonal compartments, and in axon initial segments12, 13.
Nav channels are phosphorylated by various protein kinases, leading to modulation of channel activity and cellular excitability14. A number of phosphorylation sites have been described on Nav α subunits, with no sites yet detected on Nav β subunits 14. Two-dimensional phosphopeptide mapping revealed that cAMP-dependent protein kinase (PKA) phosphorylates Ser residues S573, S610, S623 and S687 within the ID I-II linker 15. PKC phosphorylates S554, S573, S576 and S1506 16. S1506 is located in the ID III-IV linker that forms the inactivation gate 17. The calcium-dependent protein phosphatase calcineurin can dephosphorylate S573 and S623, while protein phosphatase 2A is more active at S610, and the two phosphatases are equally effective at S687 15. Increased phosphorylation is generally associated with a reduction in Nav currents, and a slowing of inactivation 18, through phosphorylation of sites in ID I-II, and ID III-IV linkers, respectively [reviewed in 14]. Phosphorylation of Nav1.2 (and Nav1.1) in Purkinje neurons also affects open channel block by β4 auxiliary subunits 19.
Nav α subunit phosphorylation may also regulate the interaction of Nav channels with proteins important in expression, localization and function. Interaction between ankyrin-G and Nav1.2 at the axon initial segment is modulated by protein kinase CK2 via phosphorylation of S1112, S1124 and S1126 in the ID II-III linker 20. Nav1.2 is also modulated by phosphorylation of Tyr residues by Src-family Tyr kinases, through multi-site phosphorylation that affects binding of Fyn to Nav1.2 via SH2 and SH3 domains, and altered inactivation due to ID III-IV linker phosphorylation 21. Nav1.1 phosphorylation has been studied to a lesser extent relative to that on Nav1.2. Nevertheless, a study of Nav1.1 and Nav1.2 expressed in Xenopus oocytes showed that current amplitudes were reduced for both ion channel subtypes after PKA phosphorylation 22. Dynamic modulation of Nav channels by phosphorylation has also been proposed to underlie changes in the biophysical and pharmacological properties of Nav currents in epileptic versus normal hippocampal neurons 23.
To better understand the complexity of Nav channel phosphorylation in mammalian brain, we used two different monoclonal antibodies (mAbs) to immunopurify Nav α subunits from rat brain. We then took an unbiased approach using nanoflow tandem mass spectrometry (nano-LC MS/MS) to systematically analyze the in vivo phosphorylation sites on the purified Nav α subunits. We found that the richness of Nav channel phosphorylation is greater than appreciated from previous studies, with fourteen in vivo phosphorylation sites identified on Nav1.2, and three on Nav1.1. These data provide novel insights into the molecular basis for modulation of Nav channel activity and cellular plasticity in mammalian brain.
We immunopurified Nav α subunits from a crude rat brain membrane (RBM) fraction prepared from freshly isolated adult whole rat brains as described 24. RBM were solubilized at 1 mg protein/ml in lysis buffer (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 10 mM sodium azide, 10 mM Tris-HCl, pH 8.0, 2 mM NaF, 1 mg/ml BSA, 1.5 μg/ml aprotinin, 10 μg/ml antipain, 10 μg/ml leupeptin, 0.1 mg/ml benzamidine, 1 mM PMSF on a tube rotator at 4°C for 30 min. The insoluble fraction was removed by centrifugation at 4°C/16,000 ×g/30 min. Two different sets of immunoaffinity purifications were performed in this study. The first employed mouse mAb K69/3, raised against a recombinant fusion protein corresponding to amino acids 1882–2005 of the cytoplasmic C-terminus of rat Nav1.2. The specificity of this mAb was previously determined by ELISA against recombinant fusion proteins corresponding to the C-termini of Nav1.1, Nav1.2, Nav1.3 and Nav1.6, and immunofluorescence staining of heterologous cells expressing Nav1.1, Nav1.2, Nav1.4 and Nav1.625. We also used mouse mAb K58/35, raised against a synthetic peptide corresponding to the highly conserved ID III-IV linker and predicted to recognize all vertebrate Nav α subunits 26. Nav α subunits were affinity-purified by incubation of 10 mg RBM lysate with 2.5 μg/ml mAb at 4°C overnight on a tube rotator. This was followed by immobilization of mAb-Nav complexes on protein-G agarose beads (500 μl of a 50% slurry) at 4°C for 2 hrs on a tube rotator. Beads were washed 6x in lysis buffer without BSA and eluted with reducing sample buffer (2% SDS, 5% 2-mercapoethanol, 10% glycerol, 62.5 mM Tris-HCL, pH 6.8) by incubation at 37°C for 15 min 10.
Immunoaffinity-purified Nav channels were size-fractionated on 6% polyacrylamide-SDS gels and visualized by staining with Coomassie G-250. The Nav α subunit band of Mr ≈260 kDa was excised, diced into small pieces and washed twice with 50% acetonitrile (ACN)/50 mM ammonium bicarbonate by vortexing for 10 min. After drying in a speed vacuum concentrator, Cys residues were reduced by incubation of the gel pieces in 10 mM DTT at 56°C for 1 hr and alkylated by incubation in 55 mM iodoacetamide at RT for 45 min. Gel pieces were then washed 10 min in 50 mM ammonium bicarbonate, dehydrated in 50% ACN/50 mM ammonium bicarbonate for 10 min and dried to complete dryness. Dried gel pieces were swollen with 100 μl of 50 mM ammonium bicarbonate containing 10 ng/μl trypsin (Promega, Madison, WI) and incubated overnight at 37°C. Digested peptide mixtures were extracted with 50% ACN/5% formic acid for 30 min, dried to complete dryness and stored at −20°C until further processing.
The peptide mixtures were either directly used for MS-analyses or further processed to enrich for phosphopeptides using PHOS-Select Iron Affinity Gel beads (P9740, Sigma-Aldrich, Saint Louis, USA). PHOS-Select beads were equilibrated by washing twice in 250 mM acetic acid/30% ACN (washing buffer). Dried peptides were resuspended in 500 μl washing buffer and immobilized on the beads by rotating at RT for 30 min. Beads were washed twice and phosphopeptides were eluted by incubation for 5 min with 500 μl 400 mM ammonium hydroxide. The eluant was dried completely and stored at −20°C until further processing.
Nav channel α subunits affinity-purified with the mAbs K69/3 and K58/35 were analyzed with an LTQ ion trap or LTQ-FT hybrid mass spectrometer (Thermo-Fisher, San Jose CA) connected to a Waters Nano-Aquity UPLC system (Waters, Milford, MA). Dried peptide samples were resuspended in 2% ACN/0.1% TFA and concentrated on a Waters Symmetry C18 280 μm × 20 mm nanoAcquity trap column at a loading flow rate of 15 μl/min. Peptides were then eluted from the trap and separated by a Waters BEH C18 1.7 μm, 100 μm × 10 cm UPLC column using a 80 minute gradient of 1–70% Buffer B (Buffer A = 0.1% formic acid, Buffer B = 95% ACN/0.1% formic acid) at a flow rate of 400 nL/min, and sprayed into a LTQ-FT ion trap mass spectrometer through a nano-electrospray source. The MS survey scan was acquired using the LTQ or FTICR mass analyzer and then the top four ions in each survey scan were subjected to automatic low energy CID for MS/MS scans.
MS/MS spectra were extracted using the program Extract msn v.4.0 of the Bioworks software v.3.3 (Thermo Finnigan, San Jose, CA, USA) with default parameters, and interpreted with Mascot v.2.2 (Matrix Science, London, UK) by searching against the SwissProt mammalian database (02/18/09; 410,518 sequences; 148,080,998 residues) and the IPI rat database (01/21/2009; 40,288 sequences; 20,569,234 residues). Individual ions scores > 31 indicate identity or extensive homology (p<0.05). Database searches were performed with a peptide mass tolerance of 20 ppm, MS/MS tolerance of 0.6 Da, strict tryptic specificity (cleavage after lysine and arginine) allowing one missed cleavage site; carbamidomethylation of Cys was set as a fixed modification, whereas oxidation (M) and phosphorylation (S, T, Y) were considered as variable modifications. Every MS/MS spectrum exhibiting possible phosphorylation was manually checked and validated based on the existence of a 98 Da mass loss (−H3PO4: phosphopeptide-specific CID neutral loss) for both precursor and fragmented ions.
Proteomic analyses of Nav α subunits immunoaffinity purified from detergent extracts of total rat brain membrane preparations were performed in this study. We followed a general approach similar to that used in our previous studies of phosphorylation of ion channels immunopurified from mammalian brain [Kv2.1 27, Kv1.2 28, BKα 29 and Kv4.2 30]. One set of analyses focused on Nav α subunits immunopurified from rat brain with the Nav1.2-specific mouse mAb K69/3, which binds at the cytoplasmic C-terminus of this α subunit (Figure 1). The other set of analyses focused on Nav channels immunopurified from rat brain with the mouse mAb K58/35, raised against the highly conserved ID III-IV linker region within Nav α subunits (Figure 1) such that it recognizes all mammalian Nav isoforms. Both mAbs were effective in affinity purification yielding amounts of Nav protein sufficient for mass spectrometry-based phospho-profiling. Immunopurification from a detergent extract obtained from 10 mg of a crude whole rat brain membrane fraction, followed by fractionation on SDS gels and Coomassie blue staining, resulted in a Nav α subunit band of Mr ≈ 260 kDa and containing approximately 1 μg of purified Nav α subunits (Figure 2A). Nav α subunit bands were excised, in-gel digested with trypsin, and the resulting tryptic peptides analyzed by nanoflow liquid chromatography tandem MS (nano-LC MS/MS) using a linear ion trap or high resolution FT-ICR mass spectrometer.
We performed sixteen independent immunopurification/nano-LC MS/MS analyses using Nav1.2-specific mAb K69/3. Nav channel α subunits contain highly conserved regions, mainly within the pseudosubunits, from which tryptic peptides exhibiting 100% identity between different Nav isoforms are obtained, and unique regions, mainly from ID linkers, from which isoform-specific tryptic peptides are obtained. The analyses of the Nav α subunit pool immunopurified with mAb K69/3 in these sixteen different experiments yielded numerous examples of peptides whose sequences were identical in more than one Nav α subunit (Table 1). In addition to these conserved peptides, a total of 929 peptides corresponding to sequences unique to a given Nav α subunit were obtained. Of these, 892 (96.0%) were specific to Nav1.2. A total of only 37 peptides across all sixteen experiments could be assigned to other Nav α subunits, the majority (20/37 or 2.1% of the peptides unique to a given Nav α subunit) corresponded to Nav1.6-specific peptides (Table 1).
These results are in sharp contrast to those obtained from nano-LC MS/MS analyses of sixteen parallel immunopurifications performed with the pan-Nav channel mAb K58/35. In this case a total of 1483 peptides unique to a given Nav channel were identified, with ≈54% from Nav1.2, ≈23% from Nav1.1, ≈15% from Nav1.6, and ≈7% from Nav1.3 (Table 1). As such mAb K69/3 represents a reliable reagent for selective immunopurification of Nav1.2 from mammalian brain, and mAb K58/35 for immunopurification of all brain Nav channel isoforms.
Based on the substantial yield of Nav1.2 from our immunoaffinity purifications, mass spectrometry analyses retrieved up to 155 Nav1.2-specific peptides in a given experiment. Mascot scores for Nav1.2 ranging from 1508 to 7609 were obtained from these sixteen independent experiments. Together, we obtained ~41% (816/2005 amino acids or a.a.) overall coverage (Figure 3) of the deduced Nav1.2 a.a. sequence (Swiss-Prot: P04775.1 or SP, identical to NCBI Reference Sequence: NP_036779.1). Among the major cytoplasmic domains, coverage was 76% for the N-terminus (96/127 a.a.), 71% for interdomain I-II (235/329 a.a.), 40% for interdomain II-III (88/219 a.a.), 62% for interdomain III-IV (32/50 a.a.) and 77% for the C-terminus (174/226 a.a.). Overall coverage within these cytoplasmic domains was ≈66% (625/951 a.a.), with many of the uncovered regions corresponding to tryptic peptides that were outside the mass range amenable to LC MS/MS analysis [between MWs of 500 and 3500 Da; 31]. Among the four pseudosubunit domains coverage was 26% for domain I (79/301 a.a.), 12% for domain II (27/231 a.a.), 23% for domain III (64/273 a.a.) and 9% for domain IV (22/249 a.a.). Overall coverage in all four pseudosubunit domains was 18% (192/1054 a.a.).
MS/MS searches against two different databases revealed proteomic evidence for expression of two different Nav1.2 isoforms in rat brain, the canonical Nav1.2 sequence (Swiss-Prot: P04775.1 or SP, identical to NCBI Reference Sequence: NP_036779.1) and a variant (IPI00476429.2, or IP) exhibiting 98.7% pairwise identity. The major difference in primary sequences is within the segment corresponding to positions 130–143 in both sequences. Peptides covering this sequence, as well as a site of an additional difference (at position 189) located within the S1 transmembrane segment within domain I could not be recovered in the tryptic peptides amenable to the MS analysis. Two peptides within both sequences show a difference in one amino acid (at positions 538 and 579), namely GFQFSLEGSR versus GFRFSLEGSR, and ASLFNFKGR versus ASLFSFKGR, in the SP versus IP Nav1.2 sequences, respectively. Interestingly, in the nano-LC MS/MS analyses matches for these two peptides were found for the IP (GFRFSLEGSR, found in 8/18 experiments; ASLFSFKGR, found in 16/18 experiments), but not the SP, version of Nav1.2 (Figure 3). Another inconsistency between these sequences is within the ID III-IV linker (a.a. 1483–1485; SP: KKMSQDIFM; IP: KKFGGQDIFM). In this case we detected the SP but not IP version of Nav1.2.
For simplicity, in spite of the fact that peptides derived from two different Nav1.2 sequences were identified in this study, all data presented here concerning amino acid positions correspond to those in the canonical Nav1.2 sequence (Swiss-Prot: P04775.1; NCBI Reference Sequence: NP_036779.1) as originally described by Numa and colleagues 32.
Immunoaffinity purification of Nav α subunits with the pan-Nav channel mAb K58/35 yielded 338 peptides specific for Nav1.1 (Swiss-Prot: P04774.1; NCBI Reference Sequence: NP_110502). Mascot scores for this protein varied between 284 and 2740 across the sixteen independent experiments. Overall sequence coverage from all sixteen MS experiments for the Nav1.1 amino acid sequence (Figure 4) was ~37% (742/2009 a.a.). Sequence coverage was 83% for the N-terminal cytoplasmic domain (100/121 a.a.), 50% for the ID I-II linker (167/337 a.a.), 31% for the ID II-III linker (69/221 a.a.), 53% for the ID III-IV linker (31/58 a.a.), and 80% for the C-terminus (180/223 a.a.). The overall coverage of these sequences within these cytoplasmic domains was 59%. Among the four pseudosubunit domains coverage was 27% for domain I (81/302 a.a.), 12% for domain II (27/230 a.a.), 24% for domain III (64/270 a.a.) and 10% for domain IV (25/249 a.a.). Overall coverage in all four Nav1.1 pseudosubunit domains was 18% (197/1051 a.a.).
We next investigated the phosphorylation status of Nav1.2 by MS analyses of tryptic peptide fragments without and with enrichment of phosphopeptides. Nano-LC MS/MS analyses led to identification of a total of 15 serine/threonine (Ser/Thr) phosphorylation sites on Nav1.2 (Figures 1, ,3).3). Of these sites, pS579 was unique to IP isoform peptides obtained from the search of the IPI rat database, the remaining 14 sites were present in both the SP and IP Nav1.2 isoforms. Two (pS468, pS610) of the 15 phosphorylation sites were only observed in experiments employing iron affinity resin for enrichment of phosphorylated peptides. Phosphorylated residues at thirteen sites were unambiguously assigned based on phosphopeptide MS/MS spectra. Among the 15 identified phospho-sites, only pS4 is located in the Nav1.2 N-terminus. Eleven phospho-sites (pS468, pS471, pS484, pS528, pS554, pS579, pS610, pS623, pS687, pS688 and pS721) are clustered on the ID I-II linker. The remaining three sites (pS1930, T1966 and S1971) are located on the cytoplasmic C-terminus (Figure 1). Figure 2B shows a representative MS/MS-spectrum for a doubly charged, singly phosphorylated Nav1.2 peptide at m/z 726.36 that was fragmented to produce a tandem mass spectrum with b- and y-ion series that describe the sequence FSpSPHQSLLSIR (a.a. 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, y92+ and y102+ fragment ions. Representative spectra for all other Nav1.2 phosphorylation sites are shown in Supplemental Figure 1, and the highest Mascot score for each site in Supplemental Figure 2. All 15 identified phosphorylation sites were from tryptic peptides unique to Nav1.2 with the exception of S610, on a tryptic peptide (DSLFVPHR) also present in Nav1.3. However, as no peptides unique to Nav1.3 were recovered from the mAb K69/3 purifications, this phosphorylation site is likely on a peptide derived from Nav1.2. A summary of phosphorylation sites identified in each of sixteen individual purifications with K69/3 is shown in Supplemental Figure 3.
Analysis of Nav α subunits after immunopurification with the pan-Nav mAb K58/35 did not reveal any additional phosphorylation sites on peptides unique to Nav1.2 (Supplemental Figure 3), but did provide corroborative evidence for phosphorylation of 10 of the 15 sites on Nav1.2 (all except pS468, pS484, pS687, pS688, and pS721; Supplemental Figure 3). Together these analyses (Supplemental Figure 3) also led to identification of three Ser phosphorylation sites within peptides unique to Nav1.1 (Figure 4). The three Ser residues could be assigned unambiguously by manual inspection of the MS/MS spectra of the corresponding tryptic peptide fragments as pS470, pS551, and pS607. Representative spectra for all three Nav1.1 phosphorylation sites are shown in Supplemental Figure 4. All three phosphorylation sites were located within the ID I-II linker. Note that pS607 was unique among all Nav1.1 and Nav1.2 sites in that it was only identified only in Experiment 12, which employed phosphopeptide enrichment (Supplemental Figure 3). Based on alignments of the Nav1.1 and Nav1.2 sequences, and similarity of surrounding amino acids, the positions of each of the Nav1.1 sites correspond to sites identified in the Nav1.2 in the ID I-II linker. They are pS470:pS471, pS551:pS554, and pS607:pS610, in Nav1.1 and Nav1.2, respectively. No phosphorylation sites were identified on the peptides unique to Nav1.6, Nav1.3 or other Nav1 channels from the Nav α subunits purified with mAb K58/35.
The rank order of isoform unique peptides purified from detergent extracts of rat brain membranes with mAb K58/35 follows the pattern of isoform expression in mammalian brain obtained from previous mRNA-based studies [Nav1.2 > Nav1.1 > Nav1.6 > Nav1.3; 6]. A previous study of the relative abundance of different Nav a subunit polypeptides in rat brain utilized subtype-specific antibodies against Nav1.1 and Nav1.2 to immunoprecipitate in vitro P-labeled Nav channels that had been purified from rat brain by classic chromatographic approaches 8. In this study, 70% of the Nav α subunits that were available for immunoprecipitation with a “pan-Nav” antibody could be immunoprecipitated with an anti-Nav1.2 antibody, and 26% with an anti-Nav1.1 antibody. This general pattern also corresponds to studies of mRNA expression levels 6.
For investigation of in vivo phosphorylation of the Nav α subunits, we undertook a proteomic analysis based on monoclonal antibody-based affinity purification of appropriately solubilized Nav α subunits combined with nano-LC MS/MS analyses. Using this approach, we identified a total of 15 Ser/Thr phospho-sites in Nav1.2 and 3 Ser phosphorylation sites in Nav1.1. Our previous studies on other ion channels immunoaffinity-purified from rat brain, namely Kv2.1 27, and BKCa channels 29, yielded a similar unexpected complexity of in vivo phosphorylation. Moreover, as we also found here, the pattern of the in vivo phosphorylation sites identified in nano-LC MS/MS analyses differed from predictions obtained from widely-used computer algorithms based on consensus sites for specific protein kinases 27, 29. These algorithms predicted a large number of phosphorylation sites on the Nav1.2 sequence (Supplemental Figure 5), but the algorithms did not consistently predict the pattern of in vivo phosphorylation detected by nano-LC MS/MS analyses of Nav1.2 α subunits purified from rat brain (Figure 5). The prediction algorithms yielded both highly ranked phosphorylation sites not detected in the in vivo situation, and poorly ranked sites that were found phosphorylated in vivo (Supplemental Figure 5). There are admittedly a number of caveats associated with mass spectrometric analysis of a sample purified from native brain 33, such as inadvertent selection of specific subpopulations of target proteins by extraction or chromatography, post facto changes in phosphorylation at specific sites, loss of specific peptides or lack of their detection in the nano-LC MS/MS analyses, etc. The actual number of phosphorylation sites present on Nav α subunits could differ from that reported here, especially if they are phosphorylated in specific brain regions that may be under-represented in our whole brain sample. Nevertheless, we suggest that the proteomic approach used here is advantageous in providing a comprehensive analysis of phosphorylation sites present in vivo on rat brain Nav channel α subunits compared to conventional consensus-site based strategies.
With respect to localization of the identified phospho-sites in the Nav1.2 primary structure, the identified pSer/pThr are located in four regions. One site (S4) is located in the cytoplasmic N-terminus. This site was not identified in consensus site prediction programs (Figure 5) and no previous studies have suggested a role for N-terminal Nav channel phosphorylation. Eleven sites are clustered in the interdomain I-II linker (sites pS468–pS721), which is known from previous studies to be the major modulatory domain of Nav channels (reviewed in 14). Within this domain, PKA has been shown to phosphorylate Nav1.2 at four sites (pS573, pS610, pS623, pS687), as determined by backphosphorylation, phosphopeptide mapping and microsequencing of Nav α subunits purified from rat brain and synthetic Nav1.2 peptides 15, 34. Sites pS610, pS623, and pS687 also score highly as PKA phosphorylation sites in consensus prediction algorithms (Figure 5). The high density of in vivo phosphorylation sites in this intracellular linker is consistent with a prominent role of this domain in phosphorylation-dependent modulation of channel gating as defined in studies on recombinant Nav1.2 α subunits expressed in heterologous cells [e.g. 35–37; reviewed in 14]. A particularly important role was defined for phosphorylation at S573, which was not detected here as an in vivo Nav1.2 phosphorylation site 37. No sites were located in the ID II-III linker, which contains three phosphorylation sites (pS1112, pS1124, pS1126) recently suggested to influence the retention of Nav1.2 at the axonal initial segment 20. Unfortunately, we were unable to recover this sequence, presumably due to the lack of tryptic cleavage sites near these sites. In the Nav1.2 C-terminus we found three novel phosphorylation sites, (pS1930, pS1971, pT1966). The C-terminus between a.a. 1890–2005 is known to be involved in the modulation of Nav1.2 function by interaction with G-protein βγ subunits 38. The novel phosphorylation sites in the Nav1.2 C-terminus could be involved in dynamic regulation of the interaction between the Nav1.2 α subunit and G-protein βγ subunit. All three of these C-terminal sites are adjacent to prolines (either Ser-Pro or Thr-Pro), raising the potential for coordinate regulation by a MAP kinase or CDK kinase family member, as has been shown for phosphorylation within the I-II linker of Nav1.6 39 and Nav1.8 40; note that analogous ID I-II sites in Nav1.2 (pS554) and Nav1.1 (pS551) are present in SerPro dipeptide motifs.
In the Nav1.1 primary structure three in vivo Ser phosphorylation sites were detected in this study, all of which were clustered in the ID I-II linker. Interestingly, all of the in vivo Nav1.1 sites identified in the ID I-II linker correspond to sites also present in Nav1.2. In each case the regions immediately adjacent to the phosphorylated residue is highly conserved, but differences within other positions within the tryptic peptides allow for unambiguous assignment of the peptide to that particular Nav α subunit. The correlation between these sites in the two different Nav α subunits may suggest that they are co-regulated by dynamic changes in phosphorylation state at these sites, as previously suggested based on consensus phosphorylation site predictions 14, and studies of recombinant Nav α subunits expressed in heterologous cells 22.
A large number of mutations in Nav1.1 and Nav1.2 have been found associated with neurological disease, particular infantile epilepsies. Over 150 mutations have been identified in patients with epilepsy 41. In primary structure, none of these mutations correspond to or are immediately adjacent to the in vivo phosphorylation sites described here. However, detailed three-dimensional structural information is not available for Nav α subunits. As such, the spatial relationships of the in vivo phosphorylation sites identified here and the locations of these debilitating mutations in the tertiary structure of Nav1.1 and Nav1.2 are not known. Recently, as a first step a solution NMR structure of the C-terminal fragment of Nav1.2 was obtained 42. Unfortunately, the Nav1.2 fragment used in these studies (1777–1882) did not contain the in vivo Nav1.2 C-terminal phosphorylation sites (pS1930, pS1966 and pS1971) identified here. Future structural studies, as well as detailed information on physiological and pathological regulation of these sites, and the effects of their mutation on expression, localization and function of Nav channels in brain neurons may help elucidate details of the roles of the in vivo phosphorylation sites described here in brain function. The powerful combination of monoclonal antibody-based immunopurification and nano-LC MS/MS approaches used here could also be used to monitor phosphorylation changes in diseased tissues using multiple reaction monitoring methods. For example, the phosphorylation sites identified here could be used in quantitative multiple reaction monitoring studies to screen for phosphopeptide biomarkers to this channel in normal and epileptic brain, where changes in Nav channel function are suggested to underlie altered neuronal excitability 43. Such approaches could lead to quantitation of the phosphorylation state of the unique and non-unique phosphopeptide ions in these and other normal and diseased tissue samples.
In conclusion, this work presents the first comprehensive MS-based analysis of phosphorylation sites on Nav α subunits purified from rat brain. Here, we used mAb-based immunoaffinity methods to purify Nav α subunits from rat brain, and identified 15 Ser/Thr in vivo phosphorylation sites on Nav1.2, and 3 in vivo Ser residues on Nav1.1. Our results demonstrate that the number of phosphorylation sites present on brain Nav1.2 and Nav1.1 in vivo greatly exceeds that suggested from previous studies, suggesting that phosphorylation of these Nav channels in rat brain provides a rich template for modifying Nav channel expression, localization and function, and cellular excitability, in response to altered physiological and pathological conditions.
This work was supported by a grant from the National Institutes of Health to JST (NS64428). We thank Brett Phinney, Rudy Alvarado and Rich Eigenheer from the UC Davis Proteomics Facility for acquiring the mass spectrometry data for this study and excellent technical advice, and Drs. Michael E. Wright and JoAnne Engebrecht for comments on this manuscript.
Supporting Information: Figures of peptide spectra for the Nav1.2 phosphopeptides, Mascot scores for the Nav1.1 and Nav1.2 phosphopeptides, summary of individual experiments, figures of peptide spectra for the Nav1.1 phosphopeptides, and results from analysis of Nav1.2 with consensus phosphorylation site prediction algorithms. This material is available free of charge via the Internet at http://pubs.acs.org.
This article references 43 other publications.