CaMKII Phosphorylation of NaV1.5 - American Chemical Society

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CaMKII phosphorylation of Na 1.5: novel in vitro sites identified by mass spectrometry and reduced S516 phosphorylation in human heart failure Anthony W Herren, Darren M. Weber, Robert R Rigor, Kenneth B Margulies, Brett S Phinney, and Donald M. Bers J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00107 • Publication Date (Web): 27 Mar 2015 Downloaded from http://pubs.acs.org on March 30, 2015

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Journal of Proteome Research

CaMKII phosphorylation of NaV1.5: novel in vitro sites identified by mass spectrometry and reduced S516 phosphorylation in human heart failure Anthony W. Herren1, Darren M. Weber1, Robert R. Rigor1, Kenneth B. Margulies2,

Brett S. Phinney1, Donald M. Bers1*

1University

2University

of California Davis, Davis, CA, USA

of Pennsylvania, Philadelphia, PA, USA

*author to whom correspondence should be addressed

KEYWORDS: CaMKII, NaV1.5, Na channel, arrhythmia, phosphorylation, mass spectrometry, cardiac, heart failure, sodium, SCN5A

Abstract

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The cardiac voltage-gated sodium channel, NaV1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, Calcium (Ca2+)/Calmodulin(CaM) dependent protein kinase II (CaMKII) has emerged as a critical regulator of NaV1.5 function through phosphorylation of multiple residues including S516, T594, and S571 and these phosphorylation events may be important for the genesis of acquired arrhythmias, as occur in heart failure. However, phosphorylation of full-length human NaV1.5 has not been systematically analyzed and NaV1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human NaV1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of NaV1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human NaV1.5.

Introduction NaV1.5 conducts Na+ current (INa) and is responsible for the initiation and maintenance of electrical propagation in the heart. Inherited mutations in the gene encoding for NaV1.5, SCN5A, have been associated with loss or gain-of-function changes in channel activity that manifest as the clinical phenotypes Brugada (BrS) or Long QT syndrome (LQTS) 1. Furthermore, NaV1.5 is posttranslationally modified (e.g. phosphorylation, methylation) and forms a macromolecular complex through association with many regulatory and accessory proteins that modulate its function and expression 2. These interactions and post-translational modifications may be important in acquired arrhythmias, such as those that occur in the context of heart failure 3. In particular, CaMKII has 2


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emerged as a key regulator of NaV1.5 through phosphorylation of the I-II linker loop (4-6, reviewed in 3). CaMKII is a basophilic protein kinase belonging to the Ca2+/CaM dependent superfamily of serine/threonine kinases. By acting as a memory molecule in response to changes in intracellular [Ca2+] resulting from high frequency, repetitive stimulation, it serves a critical role as an integrator of calcium signaling 7, 8. The cardiac isoform, CaMKIIδC, has many downstream protein targets in myocytes, including ion channels, and has been implicated in a wide range of cardiac pathologies, including arrhythmias and heart failure 8. In cardiac myocytes, phosphorylation of NaV1.5 by CaMKII shifts steady-state inactivation to hyperpolarizing potentials, enhances entry into and slows recovery from inactivation (all loss-of-function effects); and increases persistent or late INa (gain-offunction) 9. These effects mirror inherited mutations in the channel that result in “overlap” syndromes characterized by simultaneous gain and loss-of-function 10, 11. The NaV1.5 domain I-II intracellular linker loop has been shown to be a critical hotspot for CaMKII phosphorylation, including phosphorylation at S516 4, T594 4, and S571 5, 6, but the sites of phosphorylation relevant to the functional effects observed in human heart failure 12 remain controversial and additional unknown sites may exist. Although large-scale shotgun phospho-proteomic studies have identified novel sites of phosphorylation by CaMKII 13 and other kinases 14 in cardiac tissue, such studies often entirely miss low abundance protein targets, such as NaV1.5. Indeed, more targeted mass spectrometry based proteomic approaches have revealed complex patterns of phosphorylation on neuronal NaV isoforms, NaV1.1 and NaV1.2, purified from rat brain 15, as well as NaV1.5 from unstimulated mouse heart 16. To date, no proteomic studies have been done on full-length human NaV1.5 assessing global phosphorylation or phosphorylation by an individual kinase. Herein, we utilized a label-free quantitative proteomic approach to assess the global phosphorylation pattern of full length NaV1.5 expressed and purified from HEK293 cells, both at baseline and in response to in vitro phosphorylation by recombinant CaMKIIδC. We confirmed, in 3



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the human channel, several phosphorylation sites previously identified in mouse 16 and identified novel sites that may warrant functional characterization to yield further insight into Na+ channel regulation in health and disease. Additionally, peptide and spectral data presented here will facilitate future data-driven targeted proteomics approaches to identify these low-abundance phospho-peptides in human heart tissue. We also show that S516 phosphorylation is reduced in human heart failure tissue. This site is adjacent to a known methylation site 17, R513, which is important for CaMKII selectivity, and methylation at R513 decreases S516 phosphorylation in vitro 18. Taken together, this may reflect a novel reciprocal relationship between methylation and phosphorylation of some NaV1.5 CaMKII sites in heart failure, as has been similarly described for neuronal NaV1.2 purified from epileptic rat brains 19.

Materials and Methods Expression, Purification, and In Vitro Phosphorylation of NaV1.5 A mammalian DNA expression vector encoding human NaV1.5 (hNaV1.5, NCBI Reference Sequence (RefSeq) NP_932173.1), driven by a CMV promoter, was expressed in human embryonic kidney (HEK293) cells (Invitrogen #R705-07) using Lipofectamine 2000 (Invitrogen) according to manufacturer instructions at a 1:1.5 DNA (ug): Lipofectamine (ul) ratio. All DNA was sequence verified by the Sanger method before use (Retrogen, Inc.). At 48 hrs post-transfection, cells were scraped, washed with phosphate-buffered saline (PBS, 0 Ca2+, 0 Mg2+), pelleted at 2000 rcf, and lysed by two rounds of freeze-thaw fracture in liquid nitrogen. Pellets were washed in PBS solution containing (in mmol/L): 0 Ca2+, 0 Mg2+, 2 EGTA, 5 EDTA, 30 NaF, 20 sodium pyrophosphate, and 40 β-glycerophosphate at pH 7.4 to remove cytosolic proteins. Supernatants were saved for Western analysis representing the cytosolic fractions. Samples were pelleted and resuspended in membrane extraction (RIPA) buffer containing (in mmol/L): 1% sodium deoxycholate, 1% Triton X-100, 0.1% 4


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SDS, 150 NaCl, 10 NaHPO4, 1 EDTA, 1 EGTA, and 5 NaF, at pH 7.4. The resulting protein suspension was sonicated (medium intensity over 6-10 sec at a duty cycle of 35%) on ice to further solubilize protein and the remaining insoluble material was cleared by centrifugation at 15,000 rcf for 10 min. This crude membrane fraction of NaV1.5 was used for further enrichment and downstream LCMS/MS applications. The remaining insoluble fraction was resuspended in buffer containing (in mol/L): 6 urea, 0.25 sucrose, 1% CHAPS, 0.2% SDS, 0.05 Tris-Cl, 0.002 EGTA, 0.005 EDTA, 0.02 sodium pyrophosphate, 0.03 NaF, and 0.04 β-glycerophosphate at pH 7.6 and saved for Westerns. All buffers were pre-chilled to 4°C and supplemented with protease and phosphatase inhibitor cocktails (Calbiochem) at 1:10 immediately before use. Unless otherwise specified, all chemical reagents were obtained from Sigma-Aldrich. Protein concentrations of the resulting supernatants were determined by bicinchoninic acid (BCA) method (Pierce). To further enrich for NaV1.5, 600-2400 ug of crude membrane fraction was immunoprecipitated at 4°C overnight with 6-24 ug of rabbit polyclonal NaV1.5 antibody (Alomone #ASC-005, lot#2702) in immunoprecipitation (IP) buffer (in mmol/L: 150 NaCl, 10 EGTA, 10 EDTA, 10 Tris-Cl, 0.1% Triton X-100, 0.1% sodium deoxycholate, pH 7.4). This affinity purification was performed in duplicate for each sample group (n=5 sample groups) with one sample subsequently used for in vitro phosphorylation by recombinant CaMKIIδC (“CaMKII”) and the other sample serving as a baseline control (“Baseline”). Antigen/antibody complexes were captured on protein A/G magnetic beads (50ul of 50% slurry, Millipore) and washed 4X with IP buffer. With the antigen/antibody complex still bound to beads, the sample to be in vitro phosphorylated was resuspended in in vitro kinase buffer (IVK) buffer containing (in mmol/L): 50 HEPES, 100 NaCl, 10 MgCl2, 0.25 ATP, 2 CaCl2, and 0.005 CaM (Calbiochem #208690) at pH 7.2 and reacted for 10 minutes with 1 ug of purified recombinant CaMKIIδC (courtesy of Dr. Howard Schulman, Allosteros Therapeutics). In parallel, the baseline sample was incubated in IVK buffer without CaMKII and ATP. The reactions 5



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were quenched and eluted with reducing sample (RS) buffer (in mmol/L: 35 Tris-Cl (pH 6.8), 120 DTT, 4% SDS, 0.01% bromophenol blue, 16% glycerol). Samples were boiled and size fractionated by SDS-PAGE. Briefly, samples were loaded into 4-15% gradient SDS-polyacrylamide gels (TGX, BioRad) and electrophoresed at constant voltage in Tris-glycine buffer (in mmol/L: 25 Tris, 192 glycine, 0.1% SDS, pH 8.3). Gels were stained with colloidal Coomassie (AcquaStain, Bulldog Bio) to visualize NaV1.5 protein at ~220 kD and bands were manually excised for subsequent LC-MS/MS. For Western blot analysis of IPs, NaV1.5 IPs and in vitro phosphorylation reactions were performed as above with the addition of an IgG control IP (rabbit IgG antibody, Santa Cruz #sc2027). The unbound flow-through fraction of each IP was saved. Protein concentrations and volumes were standardized and 50 ug of each fraction (including the starting lysate) were loaded. For Western analysis of the lysate fractionations, 50 ug of cytosolic, membrane, and urea lysate fractions as well as non-fractionated cells lysed with RIPA buffer alone were loaded. All samples were diluted in RS buffer and equal volumes were loaded into gels for SDS-PAGE (as above). Protein was transferred to 0.2 µm PVDF membranes (Immobilon FL, Millipore) overnight at constant voltage. Membranes were washed with Tris-buffered saline supplemented with Tween-20 (TBST), blocked in milk, and probed with the following primary antibodies: for IPs, NaV1.5 at 1:250 (Alomone #ASC-013); for fractionations, NaV1.5 at 1:250 (Alomone #ASC-005) and GAPDH at 1:50,000 (Sigma #G8795). Membranes were probed with either anti-rabbit or anti-mouse 680nm or 800nm infrared fluorescent-conjugated secondary antibodies (IRDyes 800CW or 680LT, Li-Cor Biosciences) and imaged on a Li-Cor Odyssey CLx imaging station (Li-Cor Biosciences) in the 700 nm and 800 nm channels. Human Heart Tissue and S516 Phospho-antibody Generation Diseased human left ventricular tissue was obtained from the Heart Tissue Bank of the University of Pennsylvania through an IRB-approved protocol. This included 10 subjects with dilated cardiomyopathy (DCM) and 10 with ischemic cardiomyopathy (ICM). Non-failing hearts (NF; n=10) 6


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were procured from brain-dead organ donors with no history of clinical heart failure. In all cases, prospective informed consent was obtained for research use of donated heart tissue from patients or next-of-kin (for organ donors). All hearts were arrested in situ with antegrade perfusion-based cardioplegia and kept on wet ice during transportation and processing. Transmural samples of the left ventricular free wall were placed in labeled cryovials and snap frozen in liquid nitrogen. Protein from excised heart tissue was extracted and solubilized at 4°C in RIPA homogenization buffer. Briefly, frozen tissue was pulverized by mortar and pestle, and further disrupted by 3 strikes with a Polytron PT homogenizer and 3 sets of 5 strikes with a Wheaton dounce tissue grinder. Protein suspensions were cleared by centrifugation at 5000 rcf for 10 min and supernatants were analyzed by Western blot (as above). Membranes were probed with the following primary antibodies: NaV1.5 (1:350; Alomone #ASC-005), GAPDH (1:50,000; Sigma #G8795), pT286/T287 CaMKII (1:1000; Cell Signaling #3361S), and custom generated rabbit polyclonal antibodies for total CaMKIIδC (1:30,000) and phosphorylated S516 (pS516) of hNaV1.5 (1:350). Secondary antibodies were as above. Densitometry quantification was performed with ImageJ (NIH). The novel pS516 antibody was generated by inoculation of rabbits with the following synthesized peptide antigens comprising the pS516 epitope region of hNaV1.5: LTRGL(pS)RTSMKP and SLTRGL(pS)RTSMKP (21st Century Biochemicals, Inc.). Antibody was immunodepleted with the non-modified peptide SLTRGLSRTSMKP. For antibody validation, WT hNaV1.5 or nonphosphorylatable alanine mutants (generated with QuikChange II XL Site-Directed Mutagenesis, Stratagene #200522) were expressed in HEK293 cells, immunoprecipitated with an anti-pan NaV antibody (Sigma #S8809), and immunoblotted as above. IVK reactions were as described above. Dephosphorylation was performed with 10 units of Calf intestinal alkaline phosphatase (CIP, NEB #M0290S) reacted at 30°C for 30 min in the supplied buffer.

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LC-MS/MS Processing Gel bands were proteolytically digested in-gel after reduction in 10 mM DTT and alkylation in 55 mM iodoacetamide (both buffered in 50 mM ammonium bicarbonate). Sequencing-grade chymotrypsin or trypsin (250 ng, Promega) in 50 mM ammonium bicarbonate supplemented with 0.01% ProteaseMax (Promega) were used for enzymatic protein cleavage. Peptides were extracted in 60% acetonitrile/0.1% trifluoroacetic acid (TFA). Peptide solutions were lyophilized in a vacuum concentrator and reconstituted in 2% acetonitrile/0.1% TFA for LC-MS/MS. LC separation was done on a Nano Acquity (Waters) HPLC with a Proxeon nanospray source. The digested peptides were loaded onto a 100 µm x 25 mm Magic C18 100Å 5U reverse phase trap where they were desalted online before being separated using a 75 µm x 150 mm Magic C18 200Å 3U reverse phase column. Peptides were eluted using a gradient of 0.1% formic acid (A) and 100% acetonitrile (B) with a flow rate of 300 nL/min. A 60 min gradient was run with 5% to 35% B over 45 min, 35% to 80% B over 5 min, 80% B for 1 min, 80% to 5% B over 1 min, and finally held at 5% B for 8 min. Mass spectra (MS) were collected on an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) in data-dependent mode with one MS precursor scan followed by 15 MS/MS scans. A dynamic exclusion of 10 sec was used. MS spectra were acquired with a resolution of 70,000 and a target of 1 × 106 ions or a maximum injection time of 30 msec. MS/MS spectra were acquired with a resolution of 17,500 and a target of 5 × 104 ions or a maximum injection time of 50 msec. Peptide fragmentation was performed using higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) value of 27. Unassigned charge states as well as +1 and ions > +5 were excluded from MS/MS fragmentation. For validation of phosphorylation on serine 516 of NaV1.5, a ≥ 90% pure phospho-peptide was chemically synthesized (21st Century Biochemicals, Inc.) corresponding to residues SLTRGL(pS)RTSMKP of NaV1.5 with only serine 516 phosphorylated. This pure peptide was 8


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digested with chymotrypsin and analyzed by LC-MS/MS (as above). Spectra were searched and analyzed with Peaks 7 (Bioinformatics Solutions). Spectra for the resultant proteolyzed phosphopeptide TRGL(pS)RTSM were compared to the same peptide obtained from LC-MS/MS analysis of full-length, in vitro phosphorylated NaV1.5. Data Analysis Tandem mass spectra were searched and scored with both X! Tandem (The GPM, thegpm.org; version Sledgehammer 2013.09.01.2) and SEQUEST (Thermo Fisher Scientific; version 1.4.0.288) against a human database created from all protein sequences comprising the Uniprot human reference proteome database (67,085 proteins; 11/21/14), 115 protein sequences of common laboratory contaminants (http://www.thegpm.org/crap/) plus an equal number of reverse decoy sequences. Search parameters were set for a fragment ion mass tolerance of 20 PPM (or 0.02 Da for SEQUEST) and a parent ion tolerance of 20 PPM. Carbamidomethylation of cysteine was specified as a fixed modification. Deamidation of asparagine and glutamine, oxidation of methionine, dioxidation of methionine and tryptophan, acetylation of the n-terminus and phosphorylation of serine, threonine and tyrosine were specified as variable modifications. Scaffold (Proteome Software; version 4.4.1.1) was used to validate MS/MS based peptide and protein identifications. Total and exclusive peptide and spectrum counts, based on the settings below, are provided in Table S1. For identification of NaV1.5 and other NaV isoforms, protein identifications were set to achieve a protein decoy false discovery rate (FDR) ≤ 1.0% and contained at least 3 identified peptides. Peptide identifications were accepted if they could be established at ≥ 90.0% probability and achieve a peptide decoy FDR ≤ 1.0% by the Scaffold Local FDR algorithm. Actual protein and peptide decoy FDRs were 0% and this is referred to as “high stringency” in Table S1. For identification of phospho-peptides, protein identifications were set to achieve a protein decoy FDR ≤ 5.0% and contained at least 3 identified peptides. Peptide identifications were accepted if they could be established at ≥ 47.0% probability and achieve a peptide decoy FDR ≤ 9



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1.0% by the Scaffold Local FDR algorithm. Actual protein and peptide decoy FDRs were 3.8% and 1%, respectively, and this is referred to as “medium stringency” in Table S1. Protein probabilities were assigned by the Protein Prophet algorithm 20. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Only the NaV isoform protein cluster is provided in Table S1 (NaV1.1-1.9 and NaX). Total spectrum count includes the total number of (non-exclusive, non-unique) spectra associated with a particular isoform including those shared with other isoforms. Exclusive peptide or spectra are those associated only with the indicated isoform and unique refers to those that differ in sequence, charge, or modifications. Phosphorylated peptide MS/MS spectra were manually validated for the presence of a 98 Da parent ion neutral loss corresponding to the loss of H3PO4 from serine or threonine (or 80 Da from HPO3). Assignment of phosphorylation to a particular residue within a peptide sequence was done using site-discriminating ions and the calculation of an A-score (with associated probability) using the method described by Beausoleil et al. 21. For assignment of phosphorylation, spectra with < 70% peptide ID probability, no parent or ion neutral loss, and A-score probabilities < 95% were rejected. The highest scoring spectra for each peptide, based on the above criteria, were selected as representative spectra for tables and figures and taken as evidence for phosphorylation. All phosphorylation sites were identified with at least one chymotryptic or tryptic peptide ID ≥ 95%, classified as “high confidence” in Table S2 (or had reasonable evidence from a dually phosphorylated peptide). Spectra with peptide ID probabilities of 90-94% were classified as “medium confidence” and < 90% were classified as “low confidence” and are included for completeness in Table S2. Spectral counting was used to quantify the relative abundance of phospho-sites identified at baseline and with CaMKII treatment (Table S3). Specifically, sites were considered to be significant 10


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if spectral counts of assigned phospho-sites were at least two at baseline or were two-fold more abundant in CaMKII than baseline (CaMKII/Baseline ≥ 2, in Table 1). Sites that had four counts at baseline or were at least four-fold more abundant in CaMKII compared to baseline (CaMKII/Baseline ≥ 4) were assigned in Table 1. Sites that could not meet these criteria were assigned (or  if not identified) in Table 1. For phosphorylation sites with CaMKII/Baseline spectral count ratios of ≥ 2 but < 4 ( above), MS1 precursor ion total chromatographic peak area was used for further label-free quantification of the relative abundance of phospho-peptides between baseline and CaMKIIδC phosphorylated samples. Briefly, MS1 ion chromatograms were extracted, peaks picked and aligned by retention time across sample groups, and validated through a combination of manual inspection and expected isotope mass distribution ratios using Skyline 22 (MacCoss Lab, University of Washington; version 2.6). All 3 carbon isotopes were included in the calculation of total peak area: (M)+, (M+1)+, and (M+2)+. In the situation where multiple distinct phospho-peptides revealed phosphorylation at the same residue or the peptide is multiply modified, the phospho-peptide with the highest spectral count was used for quantitation. Peak areas of the same peptide were normalized across chymotryptic sample groups to the peptide MAQHDPPPWTKY, which comprises the short extracellular region spanning S1 and S2 of domain I on NaV1.5. This peptide cannot be modified by phosphorylation and was recovered in all samples at high abundance. Peak areas were averaged across sample groups (n=4). Box and whisker plots of baseline and CaMKIIδC sample peak areas were generated with Prism (GraphPad; version 5.04) and paired student’s t-test was performed. Statistical significance was taken at a p-value < 0.05. The numerical fold increases (CaMKII/Baseline) of MS1 peak areas from Figure 5 are provided in Table 1. Primary amino acid sequence alignments were performed using M-Coffee 23 by combining the output from the ClustalW, T-Coffee, and MUSCLE multiple sequence alignment methods. Where necessary, alignments were hand edited. The following NCBI (RefSeqs) were used for NaV1.5 across 11



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species: human NP_932173.1, mouse NP_067519.2, rat NP_037257.1, rabbit XP_008251214.1, dog NP_001002994.1, horse NP_001157367.1, chimp XP_001171891.2, bovine NP_776883.1, cat XP_006936590.1. For NaV1.X isoforms: NaV1.1 (human) NP_001159435.1, NaV1.1 (rat) NP_110502.1, NaV1.2 (human) NP_066287.2, NaV1.2 (rat) NP_036779.1, NaV1.3 (human) NP_008853.3, NaV1.4 (human) NP_000325.4, NaV1.5 (human) NP_932173.1, NaV1.6 (human) NP_055006.1, NaV1.7 (human) NP_002968.1, NaV1.8 (human) NP_006505.2, NaV1.9 (human) NP_054858.2. Previously identified phosphorylation and methylation sites were compiled from the literature 16, 17, 19, 24-31.

Results Expression, Purification, and In Vitro Phosphorylation of Human NaV1.5 In cardiac lysates, NaV1.5 is present as a low abundance membrane protein rendering comprehensive detection of low level phosphorylation difficult. Moreover, assignment of phosphorylation events to specific kinases in these lysates can be ambiguous. This is especially true for human donor tissue, in which experimental manipulation is often not possible. To circumvent this, we opted to express human NaV1.5 in a heterologous cell system that allowed for purification of large amounts of protein and specific manipulation with CaMKII for enhanced LC-MS/MS resolution of CaMKII mediated phosphorylation events. To date, no phospho-proteomic studies have been performed on human NaV1.5. To obtain high protein levels of NaV1.5 for LC-MS/MS, we expressed cDNA encoding for human NaV1.5 into a HEK293 heterologous cell system. These cells were lysed in a buffer series to produce the crude fractions shown in Figure 1A. Specifically, mechanical lysis by freeze-fracture in a non-detergent buffer was used to liberate cytosolic proteins (Cyto), followed by detergent solubilization to yield a crude membrane fraction (Mb), and solubilization of the detergent insoluble fraction with urea (Urea). Immunoblots of these fractions with an antibody for total NaV1.5 showed a 3-4 fold 12


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enrichment of NaV1.5 (~220 kD) in the membrane fraction versus detergent (RIPA) lysis alone without fractionation. As expected, there was no NaV1.5 present in cytosolic fractions, while the cytosolic marker GAPDH is enriched in cytosolic but not membrane fractions. Interestingly, the majority of NaV1.5 remains insoluble and is only solubilized with high molar concentrations of the chaotropic salt urea. The crude membrane fraction enriched in NaV1.5 was used for subsequent LCMS/MS analysis. NaV1.5 from membrane fractions was further enriched by a combination of immunoaffinity purification and size fractionation by SDS-PAGE following the workflow in Figure 1B. A rabbit polyclonal antibody specific to NaV1.5 was used for immunoaffinity purification, and Western blot showed that NaV1.5 protein was efficiently immunoprecipitated by the NaV1.5 but not a control IgG antibody (Figure 1C). The immunoprecipitation was ≥ 90% efficient, as NaV1.5 was not appreciably detected in the flow-through. To map CaMKII phosphorylation sites on the channel, immunoaffinity purified NaV1.5 was subjected to in vitro phosphorylation with purified, recombinant CaMKIIδC (cardiac specific isoform) while still immobilized on magnetic protein A/G beads. A parallel reaction was performed for each sample in the same buffer without the addition of CaMKII or ATP. These reactions represent “CaMKII in vitro phosphorylated” and “Baseline”, respectively, for each sample (Figure 1B). Success of the phosphorylation reaction was assessed by Western blot with a custom phosphoantibody generated against the S516 phospho-epitope of the channel (Figure 1C). S516 was more phosphorylated in CaMKII phosphorylated samples compared to baseline controls. Following IVK reactions, samples were size fractionated by SDS-PAGE, stained with coomassie and the gel bands corresponding to NaV1.5 at ~220 kD were manually excised for LC-MS/MS (Figure 1D). LC-MS/MS Analysis of Human NaV1.5 Phosphorylation Our initial testing of expressed and purified human NaV1.5 under basal conditions (without CaMKII) revealed that enzymatic cleavage with chymotrypsin provided an almost two-fold increase 13



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in sequence coverage of NaV1.5 (avg. 69%) compared with trypsin (avg. 37%). Importantly, many predicted NaV1.5 phosphorylation sites occur in regions highly dense in tryptic R and K residues, which preclude their detection when digested with trypsin because peptide fragments are below the level of detection by standard LC-MS/MS (see Figure 2, alignments Figures S1 and S2). As such, chymotrypsin was selected as the enzyme of choice for quantitative label-free MS analysis as it provided the most comprehensive coverage of potential phospho-sites. Using chymotrypsin, we consistently obtained total channel sequence coverage ≥ 65% (avg. 69%), with 97% coverage of the intracellular regions (loops I-II, II-III, III-IV, N- and C-termini; n=4 samples; n=8 technical replicates, 4 Baseline and 4 CaMKII treated; Figure 2). NaV1.5 was detected with high confidence (>90% peptide ID probability, 0% FDR) through an average of 213 exclusive unique peptides, 714 exclusive spectra, and 825 non-exclusive spectra (Table S1) across 4 samples. Surprisingly, we also confidently detected very low levels of exclusive unique peptides belonging to other (neuronal) NaV isoforms: NaV1.5 (213,714) >>> NaV1.8 (6.4, 13.8) >> NaV1.7 (1,2)/NaV1.3 (1,1) (avg. exclusive unique peptides, avg. exclusive spectrum). NaV1.8 corresponded to ~2-3% of the total NaV isoform cluster, while NaV1.5 made up ~96%. A representative MS2 chymotryptic spectrum is provided in Figure 3A. Phospho-peptides are listed in Table S2 with their corresponding masses, charge states, identification probabilities, and search algorithm scores. All phospho-sites presented were identified from at least one highconfidence chymotryptic and/or tryptic phospho-peptide. Some additional lower confidence peptides are provided where informative (Table S2). For each phospho-peptide identified, the overall highest scoring representative MS2 spectrum with a parent neutral loss of 98 Da (phosphate) is provided (Figure S3). Phospho-site localization was determined by sitediscriminating ions and assignment of an A-score provided in Tables S2 and S3 and Figure S3. Spectral counts for each phospho-site are provided in Table S3.

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Interestingly, although digestion with trypsin did not increase sequence coverage, it did provide additional and exclusive phospho-site identifications (Tables S1-S3 and Figure S3). Combined MS2 analysis revealed phosphorylation on a total of 34 residues (31 S and 3 T), summarized in Figure 2 and Table 1, the majority of which fall in the I-II loop (50%). Of the 34 sites identified, 11 were identified exclusively with chymotrypsin and 11 exclusively with trypsin. The remaining sites could be identified with either enzyme. Of particular interest is the previously identified CaMKII site S571, for which the exact localization of phosphorylation on a singly phosphorylated peptide could only be achieved with tryptic peptides (Table S2 and Figure S3). The adjacent site T570 was determined to be phosphorylated as well. Phosphorylation at S1998 and S2007 were similarly identified only in tryptic peptides but at high abundance (Table S3). These enzyme dependent differences in phospho-site identification are likely due to differences in peptide ionization and the high charge state associated with certain chymotryptic peptides. Moreover, a single spectral count of a T570/S571 dually phosphorylated chymotryptic peptide was found. Several other peptides were also found to be multiply phosphorylated (indicated in Table S2 and Figure S3), corresponding to the specific sites: S12, T17/S20, T455, S457, S460, S464, S471, S497, S528, S539, S1920/S1925, and S1934/S1937. Label-Free LC-MS/MS Relative Quantitation of CaMKII Phosphorylation Sites Twenty-one of the 34 total phospho-sites identified are completely novel, previously unrecognized sites on NaV1.5 (Table 1). Spectral counting was used to determine whether sites were of significant abundance at Baseline and in response to CaMKII treatment (see Materials and Methods, Table S3). Of the 17 sites found phosphorylated at baseline, 12 were significantly abundant (Figure 4; Table 1, Basal spectral count). Many of these sites are in good agreement with those identified previously in mouse 16 and other isoforms (Figures S1 and S2). Furthermore, many of these sites reside in or around known pathogenic inherited arrhythmia mutations (indicated in Figure S4). 15



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Twenty-three sites were found to be phosphorylated by CaMKII with significant CaMKII/Baseline ratios ≥ 2 (Figure 4; Table 1, CaMKII/Baseline spectral count). In general, CaMKII sites of high abundance (CaMKII/Baseline ≥ 4, in Table 1) were localized to small clusters on the N and C-termini and were not phosphorylated at baseline. High abundant single sites were also identified on the II-III and III-IV loops (one each). Phosphorylation of the I-II loop was more diffuse and many of these sites were also significantly phosphorylated at baseline. For example, phosphorylation at the previously identified CaMKII S516 site 4 was found at baseline and was increased by CaMKII. To better confirm the determination of CaMKII phosphorylation on sites within the I-II loop (with CaMKII/Baseline ratios ≤ 4, in Table 1), a label-free approach was developed comparing the MS1 precursor ions of chymotryptic phospho-peptides across four individual sample sets (Figure 5). Peak areas were quantified, averaged and normalized for comparison of relative abundance between baseline and CaMKII treated groups (representative comparison for pS516 in Figure 3C). A statistically significant fold increase in MS1 peak area (Table 1) was found for 6 of the 8 sites assessed and taken as evidence for CaMKII phosphorylation of one or more of the S/T residues contained in the respective peptides. Quantification of the phospho-peptides for the high basal sites S497/S499 and the high abundant CaMKII sites S1934/S1937 are provided as negative and positive controls (Figure 5). Peak areas for tryptic phospho-peptides were not determined. Although CaMKII phosphorylation can only be ascribed for an entire peptide and not individual sites, the lack of phosphorylation assignment for other S/T residues contained within a peptide was taken as good evidence that those sites do not contribute to changes in peak area of that peptide. Interestingly, several sites within the I-II loop were highly phosphorylated at baseline and some had statistically significant increases with CaMKII treatment (Figure 4 and Table 1). Sites within the N- and C-terminus had much larger fold increases with CaMKII treatment (ratios ≥ 4) and had no or low phosphorylation at baseline. For example, S1925 in the C-terminus (Figure 3A) and S11/S12 in the N-terminus are novel sites that were not phosphorylated at baseline but were highly 16


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phosphorylated with CaMKII treatment. Interestingly, S1925 and S1920 are novel sites that are within the well-known CaM binding IQ motif in the NaV1.5 C-terminus (E1901-Q1930, Figure 2). The adjacent sites S1934 and S1937 are also phosphorylated by CaMKII. Furthermore, CaMKII sites were found at S1865 and S1885, which border an EF hand motif just upstream of the IQ domain (E1773-D1852, Figure 2). S1503, the only phospho-site identified in the III-IV linker (known to be important for INa inactivation), was found to be phosphorylated in 3 unique peptides (Table S2). These peptides are highly phosphorylated by CaMKII and not detected at baseline (Figure 4 and Table 1). Importantly, these peptides have high sequence conservation across all 9 NaV1.X channel isoforms (some at 100%, Figure S2). Since exclusive peptides from other channel isoforms were definitively found in HEK293 cells at very low levels (ex. NaV1.8), the localization of S1503 phosphorylation to any single isoform was not possible. S1503 phosphorylated peptides may have been contributed by both NaV1.5 and/or other NaV isoforms. Of the CaMKII sites identified, several exist in non-canonical consensus sequences that deviate from the traditional RXXS/T CaMKII consensus (Figure 2 and S1). In particular, the consensus R, while preferred at the P-3 position, is tolerated anywhere from P-3 to P-8 in the phospho-peptides identified, similar to that shown with computational surface mapping of consensus sites 32. Movement of the consensus arginine likely influences the binding affinity and thus efficiency of phosphorylation on a particular site. CaMKII Phosphorylation of S516 In Vitro and in Human Heart Failure Phosphorylation of the previously identified CaMKII site S516 was confirmed by LC-MS/MS in both chymotryptic and tryptic phospho-peptides (Table S2). However, these peptides fragmented poorly, probably due to the labile nature of phosphate groups and the specific primary backbone sequence surrounding this site. As such, we digested a synthetically produced S516 phosphopeptide with chymotrypsin to yield the peptide TRGLSRTSM, which we analyzed by LC-MS/MS and 17



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compared with the same phospho-peptide identified from full-length, CaMKII treated channel. The peptide spectra, compared in Figure 3B, are identical. Because we have previously assigned biophysical function to S516 4 and CaMKII is known to be more abundant and more active in HF 33, 34, we sought to determine the phosphorylation status of S516 in failing and non-failing human donor hearts. To this end, we generated a custom phosphospecific rabbit polyclonal antibody against the phosphorylated S516 epitope of NaV1.5. Antibody phospho- and site-specificity were determined with in vitro kinase assays (with CaMKIIδC as above) using WT or phospho-site mutant NaV1.5 immunoprecipitated from HEK293 cell lysates. Figure 6A shows that the pS516 antibody detects phosphorylation at baseline that is increased with CaMKII treatment, decreased with phosphatase (CIP) treatment, and is entirely abolished with specific mutation of S516 to non-phosphorylatable alanine (but not by mutation of other S/T within the I-II or II-III loop). This antibody was used to probe phosphorylation in human ventricular myocardium homogenates from non-failing (NF), ischemic cardiomyopathy (ICM), and dilated cardiomyopathy patients (DCM; Figure 6B, n=10 for each group). Surprisingly, when normalized to total NaV1.5 channel, S516 phosphorylation was significantly decreased in human ICM and DCM compared to non-failing donor controls (Figure 6C). As previously reported 34, phosphorylation at the CaMKII T287 autophosphorylation site was significantly increased in disease samples (Figure 6C). As discussed below, this might be due to a reciprocal interaction between increased methylation and decreased phosphorylation at R513/S516 and possibly other sites in heart failure. This is the first demonstration of a decrease in phosphorylation at any site for NaV1.5 in heart failure.

Discussion Previous mass spectrometry-based studies have shown that the rat neuronal NaV1.1 and NaV1.2 channels 24, as well as NaV1.5 from mouse 16, are multi-phosphorylated. As such, phosphorylation 18


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across multiple sites may act synergistically, additively, or antagonistically to produce graded effects on channel gating, as has been shown for other channel proteins (e.g. potassium channels) 15, 35.

Multi-site graded regulation would allow for fine-tuning of channel function in response to

diverse stimuli and a graded response, unlike an all-or-none response, widens the physiological dynamic range within which channels can respond. For the first time, we now extend these findings to human NaV1.5 and further delineate CaMKII phosphorylation of the channel. Few mass spectrometry based studies of CaMKII specific phosphorylation have been reported. Of note, the Heck group reported a label-based phosphoproteomics study assaying global CaMKII phosphorylation in mouse with and without CaMKII inhibition 13 and another study assayed CaMKII specific phosphorylation of the CX43 C-terminus 36. Unfortunately, the low abundance of NaV1.5 (and other ion channel membrane proteins) in complex cardiac protein lysates, such as in the Heck study 13, precluded its detection without enrichment and NaV1.5 phosphorylation was not found. A more targeted approach for low abundance ion channel membrane proteins using in vitro purified and phosphorylated protein, such as that performed for CaMKII phosphorylation of CX43 36, PKC phosphorylation of troponin 37 or cMyBP-C 38,

allows for enhanced detection of phospho-peptides. Here, we have combined purification of full-

length NaV1.5 with in vitro phosphorylation by recombinant CaMKII and quantified relative phospho-peptide abundance using a label-free mass spectrometry based approach. Indeed, our results show that NaV1.5 is multi-phosphorylated and complex patterns of phosphorylation by CaMKII and other kinases likely contribute to graded channel regulation. The study by Marionneau et al. 16 provided an initial baseline of multi-site phosphorylation of NaV1.5 in unstimulated mouse hearts. However, while there is high primary amino acid sequence conservation within the transmembrane domains of the NaV family across isoform and species, there is considerable variation within the intracellular domains (see Figures S1 and S2), especially in regions containing kinase consensus motifs. Other studies have shown similar poor evolutionary 19



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conservation of phosphorylation sites in unstructured regions 39, 40. In fact, the NaV1.4 I-II loop is much shorter and lacking in phosphorylatable sites compared to its NaV1.5 counterpart (Figure S2). As such, specific phosphorylation events may differ in important ways across species and isoforms. Indeed, from an evolutionary perspective, this may serve to confer species and isoform specific differences in channel behavior and function. In Figure 2 and Table 1, we report the first comprehensive analysis of phosphorylation on human NaV1.5. Furthermore, many basophilic phosphorylation sites occur in basic regions rich in R and K residues that cannot be resolved with short tryptic peptides. We have circumvented this problem with the use of chymotrypsin and improved total sequence coverage by more than double compared to previous studies. Moreover, we distinguish sites phosphorylated specifically by CaMKII at high abundance. Interestingly, we found CaMKII phosphorylation to be more concentrated in structured regions of the channel, such as the C-terminus, and more distributive in disordered regions such as the I-II loop. Many of the phosphorylation sites we identified are in good agreement with conserved sites similarly identified in mouse NaV1.5 or other NaV isoforms (Figures S1 and S2). Additionally, many inherited arrhythmia mutations causing BrS and/or LQTS occur in or around some of the phosphorylation sites identified here (Figure S4). A mutation in a phosphorylation consensus may alter a site’s ability to be phosphorylated or transmit a functional response. This may provide clues for the effects of these phosphorylation sites and/or inherited mutations on INa and further suggests these phosphorylation sites are important in vivo. Phosphorylation of the I-II Loop CaMKII phosphorylation of S571 is reported to shift the voltage dependence of channel steady state inactivation to hyperpolarizing potentials, enhance entry into and recovery from inactivation, and increase late INa 5, 6. Although CaMKII specific phosphorylation at this site was not determined, we confirmed this site as phosphorylated in human NaV1.5 even at baseline. Interestingly, the S571 kinase consensus motif, RRTS, is a preferred and highly efficient substrate for PKA 41. R at the P-2 20


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position is significantly disfavored for phosphorylation by CaMKII but highly favored by PKA 32. We also identified novel phosphorylation of the adjacent T570 site, which also occurs at baseline, and we recovered a peptide phosphorylated at both T570 and S571. Thus, this consensus region may allow for promiscuous phosphorylation by multiple kinases (e.g. PKA and CaMKII) and serve as an important integrator of multiple signaling pathways. Nevertheless, the increase in phosphorylation observed at S571 in mouse 42, dog and human heart failure 6 is inconsistent with the NaV1.5 channel biophysical effects reported in patch clamp studies of ventricular myocytes from failing hearts 12 (for review see 3). Increased phosphorylation at S571 would be expected to negatively shift the steady state inactivation curve 5, 6, but the most consistent results of INa in failing hearts report no change in steady state inactivation or activation, a decrease (or no change) in INa density, and an increase (or no change) in late INa 3. Furthermore, we previously reported a nonfunctional effect for S571E phospho-mimetic mutants expressed in HEK293 cells 4. Thus, although S571 is clearly phosphorylated, the kinase involved and functional effects on INa remain controversial. CaMKII also phosphorylates S516 within the I-II loop 4, which reduces channel availability and favors entry into intermediate inactivation. We show here that this site is phosphorylated at baseline and phosphorylation can be furthered by CaMKII. S516 phosphorylation was detected in both LC-MS/MS studies of human channel in vitro and in Western blot of human heart tissue. This site is particularly interesting because its human kinase consensus, RGLS, is poorly conserved across species (Figure S1). For example, the critical R at P-3 is replaced by an H in rodent NaV1.5 and therefore is not a CaMKII consensus sequence. Interestingly, a similar RXXS CaMKII consensus motif is found in the sequence surrounding S516 for several species, including rabbit and dog (yellow, Figure S1). Conserved and similar motifs can also be found in certain other NaV isoforms, such as a known p38 MAP kinase site at S553 in NaV1.6 31 and a reciprocally regulated methylation/phosphorylation consensus at R563-S568 in NaV1.2 (see Figure S2). This highlights 21



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the importance for studying phosphorylation in the context of species and isoform. Furthermore, the majority of phosphorylation sites we identified (50%) reside in the relatively disordered NaV1.5 I-II loop and are diffuse and distributive in this region. This suggests that phosphorylation within the I-II loop may be permissive and phosphorylation of specific sites may be less important for INa effects than the general location and number of sites (allowing for graded channel regulation across multiple sites). Moreover, the key CaMKII consensus R513 at P-3 (as well as R526 and R680) has been shown to be methylated in mass spectrometry studies of human NaV1.5 in vitro 17. Follow-up work in human HF found elevated methylation at R526 43. Further studies revealed that methylation of R513, R526, and R680 act in concert to promote an increase in channel trafficking to the membrane 44 and that methylation of R513 directly decreases phosphorylation of S516 in vitro 18. This is consistent with recent work demonstrating a reciprocal relationship between phosphorylation and methylation of neuronal NaV channels 19. Taken together, the above studies lay the groundwork for understanding our surprising observation that S516 phosphorylation is reduced in human HF, despite an increase in CaMKII activation that is typical in human HF 34 (Figure 6). However, we cannot fully exclude the possibility that the affinity of our S516 phospho-antibody is affected by methylation at R513. Thus, while there might be enhanced local phosphatase activity 45, 46 that limits phosphorylation at S516, as observed for several ion channel 47, 48 and contractile proteins in HF 38, 49, 50, methylation at R513 could also inhibit CaMKII-dependent phosphorylation at S516. Whether this observation is unique to S516 or a general feature of the I-II loop is a topic for future investigation. Limiting phosphorylation at S516 may function to blunt graded regulation of INa inactivation gating and/or alter channel expression through reciprocal crosstalk with methylation. The decreased S516 phosphorylation observed here is consistent with the lack of change in INa inactivation seen in failing human heart myocytes 12. However, because CaMKII-dependent NaV1.5 phosphorylation promotes late INa 9 and increased late INa is also characteristic of the HF phenotype 22


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12, 42,

phosphorylation at some of the other high abundance sites identified here (Table 1) may be

responsible. We also previously identified in vitro CaMKII phosphorylation of NaV1.5 at T594 4. We could not confirm phosphorylation at this site by LC-MS/MS, which may be due to technical limitations associated with the fragmentation and detection of peptides containing this site. Phosphorylation of the N-terminus CaMKII phosphorylated S11/S12 and S42 within the N-terminus and neither site is phosphorylated at baseline. Both sites are highly conserved across species and isoform. S11 and S12 are novel previously unreported phospho-sites and S42 phosphorylation is now confirmed in human, having been similarly identified in NaV1.5 from mouse heart 16. We can now also ascribe S42 as a target of CaMKII. The functional significance of phosphorylation at S11/S12 and S42 are unknown but previous studies have implicated the N-terminus and/or other regions in α−α subunit interactions 51. S11/S12 fit within a traditional CaMKII consensus 41, which is especially favored because of the presence of a hydrophobic F at the P+1 position 41. S42, on the other hand, is in a non-traditional CaMKII consensus with the critical basic R at the P-8 position instead of P-3 position. The N- and Ctermini of NaV1.5 are less disordered than the I-II loop and local primary and secondary sequence can significantly impact the efficiency of phosphorylation at a site and allow for phosphorylation of non-traditional consensus motifs 52, 53. Higher order secondary and tertiary structures are of particular interest as backbone chain folding may generate a traditional consensus from nontraditional sequence 52. This is reiterated by the poor predictive ability of in silico phosphorylation consensus algorithms and emphasizes the need to assess phosphorylation of whole protein empirically 53. Phosphorylation of the C-terminus

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The C-terminus of NaV1.5 is also heavily phosphorylated by CaMKII at several closely clustered sites, including S1920, S1925, S1934, S1937 and S1998. Just upstream, another cluster, including S1865 and S1885, is also phosphorylated. Similar to sites in the N-terminus, these sites were not phosphorylated at baseline, are present in structured, conserved domains, and many were found to be multiply phosphorylated. All of these sites are novel with unknown function and are conserved across species and isoforms. A recent abstract proposed that some of these sites are phosphorylated in mice 54. Interestingly, S1920 and S1925 lie within (and S1934 and S1937 are directly adjacent to) the well-described CaM binding IQ motif, which has complex effects on channel gating 3. S1865 and S1885 reside within the NaV1.5 EF hand domain. This region of the C-terminus is also known to be a hot zone for interaction with many accessory proteins that may modulate channel function 2. It is interesting to speculate that phosphorylation at one or more of these sites may alter CaM binding affinity or protein partner interactions (as suggested for the type I Na+/H+ exchanger

55),

and perhaps contribute to altered INa gating.

Phosphorylation of the II-III and III-IV Loops We identified another novel CaMKII phosphorylation site at S1503 in the III-IV loop. This 42 amino acid loop is crucial for NaV fast inactivation gating via the IFM motif. As such, this site is 100% conserved across species and isoform. S1503 is just 16 amino acids directly downstream of the IFM and phosphorylation at this site has only been indirectly inferred from in vitro experiments of rat NaV1.5 channels phosphorylated by PKC 27. We show here that S1503 is also phosphorylated by CaMKII and provide the first MS-based evidence for phosphorylation of the III-IV loop in a member of the NaV family. Our highly sensitive LC-MS/MS analysis also revealed the presence of neuronal NaV channels in HEK293 cells (at very low abundance). All phosphorylation sites in this study could be attributed exclusively to NaV1.5 except S1503, which may have been contributed by NaV1.5, neuronal isoforms, or both. The presence of neuronal NaV isoforms could complicate INa recordings in HEK293 cells, but 24


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INa function is likely dominated by the overexpressed channel. Low abundant neuronal NaV isoforms in cardiomyocytes have been suggested to contribute to late INa 56, 57, especially NaV1.8 58. Further investigations of this novel CaMKII phosphorylation site in NaV1.5 and other channel isoforms is warranted. Moreover, previous studies have shown evidence for a direct interaction between the III-IV loop and the C-terminus either directly or indirectly via CaM (reviewed in 3). It is possible that a complex combination of phosphorylation at S1503 and/or sites within the C-terminus allow for graded regulation of fast inactivation gating. Lastly, we identified the first phosphorylation site on a NaV channel within the II-III loop. This site was phosphorylated at baseline and by CaMKII. The II-III loop has an important role in channel coupling to the cytoskeleton as evidenced by its interaction with the Ankyrin-G/β-spectrin complex 5, 59

and this site may serve a role in modulating protein partner binding interactions.

In summary, S516 phosphorylation is reduced in human HF and LC-MS/MS has revealed multiple CaMKII phosphorylation sites on human NaV1.5. In addition, the novel sites we identified here will facilitate further functional studies to better understand some of the NaV1.5 gating phenomena observed in cardiac myocytes. How these phosphorylation events influence channel conformation to alter function and how they are temporally regulated remains to be determined. Functional studies will be needed, both in simplified cell systems and in native myocytes. However, some effects may be due to multi-site, graded phosphorylation across several sites. Additionally, this work has broader implications for the study of neuronal NaV channels that are involved in excitatory disorders (e.g. epilepsy, pain) similar to arrhythmias in heart. This study is a significant step forward in our understanding of channel regulation and provides a phospho-proteomic map to guide further exploration.

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Figure 1. Purification of NaV1.5 for LC-MS/MS analysis. (A) Biochemical fractionations of NaV1.5 expressing HEK293 cells (4 fractions from n=2 representative samples: Cyto (two consecutive cytoplasmic washes), Mb (membrane), and Urea (detergent insoluble). An equal amount of a nonfractionated whole-cell lysate (RIPA) is provided for comparison. Membranes were probed for NaV1.5 and GAPDH. (B) Proteomics workflow for identification and label free quantification of in vitro CaMKII phosphorylation sites on human NaV1.5. (C) NaV1.5 immunoprecipitated from crude Mb (membrane) fractionations with either NaV1.5 or control IgG antibody, reacted with or without CaMKII, and probed with pS516 or total NaV1.5 antibody. Starting lysates and flow-throughs are

26


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shown for comparison. (D) Representative full 1-dimensional SDS-PAGE gel of immunopurified NaV1.5. NaV1.5 gel bands running at ~220 kDa (boxed in red) were cut out for LC-MS/MS.

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1 MANFLLPRGT SSFRRFTRES LAAIEKRMAE KQARGSTTLQ ESREGLPEEE APRPQLDLQA N terminus 101 TIFRFSATNA LYVLSPFHPI RRAAVKILVH SLFNMLIMCT ILTNCVFMAQ HDPPPWTKYV IS1 201 IIMAYTTEFV DLGNVSALRT FRVLRALKTI SVISGLKTIV GALIQSVKKL ADVMVLTVFC IS4 301 WESLDLYLSD PENYLLKNGT SDVLLCGNSS DAGTCPEGYR CLKAGENPDH GYTSFDSFAW 401 SFYLVNLILA VVAMAYEEQN QATIAETEEK EKRFQEAMEM 501 DGPRAMNHLS LTRGLSRTSM KPRSSRGSIF TFRRRDLGSE 601 VSLLGAGDPE ATSPGSHLLR PVMLEHPPDT TTPSEEPGGP 701 PLWMSIKQGV KLVVMDPFTD LTITMCIVLN IIS1 801 MSNLSVLRSF RLLRVFKLAK SWPTLNTLIK IIS4 901 ETMWDCMEVS GQSLCLLVFL LVMVIGNLVV IIS6 1001 LPSCIATPYS PPPPETEKVP PTRKETRFEE

TLFMALEHYN IIGNSVGALG LNLFLALLLS GEQPGQGTPG

1101 TASSEAEASA SQADWRQQWK AEPQAPGCGE TPEDSCSEGS 1201 IVEHSWFETF IIFMILLSSG ALAFEDIYLE ERKTIKVLLE IIIS1 1301 SLRTLRALRP LRALSRFEGM RVVVNALVGA IPSIMNVLLV IIIS4 1401 NFDNVGAGYL ALLQVATFKG WMDIMYAAVD SRGYEEQPQW 1501 LGSKKPQKPI PRPLNKYQGF IFDIVTKQAF DVTIMFLICL IVS1 1601 LSIVGTVLSD IIQKYFFSPT LFRVIRLARI GRILRLIRGA IVS4 1701 MLCLFQITTS AGWDGLLSPI LNTGPPYCDP TLPNSNGSRG 1801 FDPEATQFIE YSVLSDFADA LSEPLRIAKP NQISLINMDL 1901 EEVSAMVIQR AFRRHLLQRS LKHASFLFRQ QAGSGLSEED IQ Motif 2001 LADFPPSPDR DRESIV

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SKKLPDLYGN PPQELIGEPL EDLDPFYSTQ KTFIVLNKGK

EYTFTAIYTF ESLVKILARG FCLHAFTFLR DPWNWLDFSV IS2 IS3 LSVFALIGLQ LFMGNLRHKC VRNFTALNGT NGSVEADGLV IS5 AFLALFRLMT QDCWERLYQQ TLRSAGKIYM IFFMLVIFLG IS6 LKKEHEALTI RGVDTVSRSS LEMSPLAPVN SHERRSKRRK RMSSGTEECG EDRLPKSDSE I-II Loop ADFADDENST AGESESHHTS LLVPWPLRRT SAQGQPSPGT SAPGHALHGK KNSTVDCNGV I-II Loop QMLTSQAPCV DGFEEPGARQ RALSAVSVLT SALEELEESR HKCPPCWNRL AQRYLIWECC I-II Loop MTSEFEEMLQ VGNLVFTGIF TAEMTFKIIA LDPYYYFQQG WNIFDSIIVI LSLMELGLSR IIS2 IIS3 NLTLVLAIIV FIFAVVGMQL FGKNYSELRD SDSGLLPRWH MMDFFHAFLI IFRILCGEWI IIS5 SFSADNLTAP DEDREMNNLQ LALARIQRGL RFVKRTTWDF CCGLLRQRPQ KPAALAAQGQ II-III Loop DPEPVCVPIA VAESDTDDQE EDEENSLGTE EESSKQQESQ PVSGGPEAPP DSRTWSQVSA II-III Loop TADMTNTAEL LEQIPDLGQD VKDPEDCFTE GCVRRCPCCA VDTTQAPGKV WWRLRKTCYH II-III Loop YADKMFTYVF VLEMLLKWVA YGFKKYFTNA WCWLDFLIVD VSLVSLVANT LGFAEMGPIK IIIS2 IIIS3 CLIFWLIFSI MGVNLFAGKF GRCINQTEGD LPLNYTIVNN KSQCESLNLT GELYWTKVKV IIIS5 EYNLYMYIYF VIFIIFGSFF TLNLFIGVII DNFNQQKKKL GGQDIFMTEE QKKYYNAMKK IIIS6 III-IV Loop NMVTMMVETD DQSPEKINIL AKINLLFVAI FTGECIVKLA ALRHYYFTNS WNIFDFVVVI IVS2 IVS3 KGIRTLLFAL MMSLPALFNI GLLLFLVMFI YSIFGMANFA YVKWEAGIDD MFNFQTFANS IVS5 DCGSPAVGIL FFTTYIIISF LIVVNMYIAI ILENFSVATE ESTEPLSEDD FDMFYEIWEK IVS6 PMVSGDRIHC MDILFAFTKR VLGESGEMDA LKIQMEEKFM AANPSKISYE PITTTLRRKH C terminus APEREGLIAY VMSENFSRPL GPPSSSSISS TSFPPSYDSV TRATSDNLQV RGSDYSHSED C terminus

Figure 2. LC-MS/MS primary sequence coverage for human NaV1.5. Combined total primary sequence coverage (yellow) of 82% for NaV1.5 compiled from chymotryptic and tryptic peptides recovered by LC-MS/MS (n=5 paired samples). Chymotrypsin consistently yielded coverage ≥ 65%. Transmembrane domains and intracellular motifs are underlined. All phosphorylation sites identified in this study are in red. Primary protein sequence is NCBI RefSeq NP_932173.1.

28


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Figure 3. NaV1.5 phosphorylated peptide spectra identified by LC-MS/MS. (A) Representative MS2 spectrum for pS1925 with ions (y=blue, b=red) and neutral losses (green) labeled. Site localization was determined with the assignment of an A-score based on site-discriminating ions. (B) A synthesized peptide (inverted, red) corresponding to phosphorylated S516 is identical to the same peptide resolved by LC-MS/MS analysis of full-length NaV1.5. (C) Representative ion chromatograms for baseline and CaMKII MS1 parent precursors aligned by retention time. Total MS1 peak areas were used for label free quantification of relative abundance.

29



I-II

N-term

NaV1.5 Domain

C-term

II-III III-IV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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S11 S12 T17 S20 S42 S61 T455 S457 S460 S464 S471 S483 S484 S497 S499 S510 S516 S528 S539 T570 S571 S577 S664 S667 S1003 S1503 S1865 S1885 S1920 S1925 S1934 S1937 S1998 S2007

Baseline CaMKII

0

5

10

15

20

25

30

70

100

Total Spectral Count Figure 4. CaMKIIδ δC phosphorylates NaV1.5 at multiple novel sites in vitro. Total spectral count as a measure of phospho-peptide abundance for baseline and CaMKII treated NaV1.5 (from n=5 paired samples). All sites included in counts were identified with an A-score probability ≥ 95% based on site-discriminating ions (Table S3). Abundant phosphorylation sites occur in tight clusters on the N- and C-termini. The II-III and III-IV loops each contain one abundant, novel site. Phosphorylation of the I-II loop is diffuse and includes both novel and previously identified sites (S516, S571). 30


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Figure 5. CaMKII increases phosphorylation of sites within the I-II loop above baseline. Label-free comparison of indicated chymotryptic phospho-peptide peak areas between baseline and CaMKII treatment (n=4 each peptide; * = p-value < 0.05, ** = < 0.01, *** = < 0.001, vs. Baseline). Phospho-peptides used for quantification were the most abundant phosphorylated peptides corresponding to confident identifications of the indicated phosphorylation sites. Fold increases provided in Table 1. The high abundant phospho-peptides corresponding to phosphorylation at S497/S499 and S1934/S1937 are provided for comparison.

31



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Figure 6. S516 phosphorylation is decreased in human heart failure. (A) A novel phospho-site antibody generated against phosphorylated S516 of human NaV1.5 is phospho- and site-specific. Phosphorylation at S516 is decreased by treatment with CIP (calf-intestinal phosphatase), increased above baseline with CaMKIIδC, and absent with mutation of S516 to nonphosphorylatable alanine (but not other site mutants). (B) Representative immunoblots of cardiac homogenates from donor non-failing (NF) and ischemic (ICM) or dilated (DCM) failing human 32


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hearts. Quantified in (C). Phosphorylated S516 of NaV1.5 or T287 of CaMKII relative to total and normalized to NF. S516 phosphorylation is decreased. Autophosphorylation of T287 on CaMKII is increased (n=10 each group; * = p-value < 0.05).

33



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Table 1. Summary of phosphorylation sites identified by LC-MS/MS

Site S11 S12 T17 S20 S42 S61 T455 S457 S460 S464 S471 S483 S484 S497 S499 S510 S516 S528 S539 T570 S571

CaMKII/ Base Spectral Count a             Baseline Spectral Count a

S577 S664 S667  S1003  S1503  S1865

Peak Area (Fold ) b

Basal vs. CaMKII CaMKII CaMKII

ND 7.9 7.9 9.8

CaMKII Basal, CaMKII CaMKII CaMKII

9.8

Basal, CaMKII

ND

3.5 3.5 ND 1.2 (none) ND

CaMKII Basal Basal, CaMKII Basal Basal Basal, CaMKII CaMKII CaMKII Basal Basal, CaMKII

1.2 (none)

Basal

ND

CaMKII CaMKII CaMKII CaMKII

Previous Kinase Assoc. SGK CaMKII PKA CaMKII SGK PKC -

Previous Identification (Novelty)/Functional Role Novel Novel Novel Novel LC-MS/MS; mouse; basal c Novel Novel LC-MS/MS; mouse; basal c LC-MS/MS; mouse; basal c Novel Novel LC-MS/MS; mouse; basal c LC-MS/MS; mouse; basal c LC-MS/MS; mouse; basal c Novel LC-MS/MS; mouse; basal c In vitro; human; ( - ) shift inactivation d In vitro; trafficking & INa e Novel Novel In vitro/ex vivo; human, dog, mouse; ( - ) shift inactivation and late INa f Novel LC-MS/MS; mouse; basal c LC-MS/MS; mouse; basal c Novel In vitro; rat; ( - ) shift inactivation, INa g Novel

a

Derived from spectral counts provided in Table S3, Supporting Information. Base or CaMKII/Base ≥ 4 = X4; ≥ 2 = X2; < 2 = ; 0 = . See Materials and Methods for more detailed explanations. b MS1 peak area fold increases from Figure 5. Calculated for sites with CaMKII/Base ≥ 2 but < 4. ND = not determined. c (16) d (4) e (28) f (5), (6) g (27) 34


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 S1885 S1920  S1925  S1934 S1937  S1998 S2007

CaMKII CaMKII CaMKII CaMKII Basal, CaMKII CaMKII Basal

-

Novel Novel Novel Novel Novel Novel Novel

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ASSOCIATED CONTENT Supporting Information Sequence alignments of NaV1.5 across species (Figure S1) and across isoform (Figure S2), peptide and spectral counts for NaV isoforms (Table S1), hNaV1.5 chymotryptic and tryptic phosphopeptides identified by LC-MS/MS with associated scores (Table S2), hNaV1.5 chymotryptic and tryptic MS2 phospho-spectra identified by LC-MS/MS (Figure S3), hNaV1.5 phospho-site spectral counts and associated scores (Table S3), inherited arrhythmia mutations in or around phosphorylation sites (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Donald M. Bers, Ph.D. Silva Chair for Cardiovascular Research Distinguished Professor & Chair Department of Pharmacology University of California, Davis Genome Building Rm 3513 Davis, CA 95616-8636 Ph: 530-752-6517 Fax: 530-752-7710 36


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E-mail: [email protected] Funding Sources This study was supported by the National Institutes of Health grants P01-HL-080101, R01-HL105242, and training grant T32-GM-099608 and the Fondation Leducq Transatlantic CaMKII Alliance. ACKNOWLEDGEMENT The authors thank Céline Marionneau (Université de Nantes, Nantes, France) for helpful discussions in the early stages of this work.

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ABSTRACT GRAPHIC

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CaMKII Phosphorylation of NaV1.5 - American Chemical Society

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