Trafficking-dependent phosphorylation of Kv1.2 regulates voltage-gated potassium channel cell surface expression

Abstract

Kv1.2 alpha-subunits are components of low-threshold, rapidly activating voltage-gated potassium (Kv) channels in mammalian neurons. Expression and localization of Kv channels is regulated by trafficking signals encoded in their primary structure. Kv1.2 is unique in lacking strong trafficking signals and in exhibiting dramatic cell-specific differences in trafficking, which is suggestive of conditional trafficking signals. Here we show that a cluster of cytoplasmic C-terminal phosphorylation sites regulates Kv1.2 trafficking. Using tandem MS to analyze Kv1.2 purified from rat, human, and mouse brain, we identified in each sample in vivo phosphoserine (pS) phosphorylation sites at pS434, pS440, and pS441, as well as doubly phosphorylated pS440/pS441. We also found these sites, as well as pS449, on recombinant Kv1.2 expressed in heterologous cells. We found that phosphorylation at pS440/pS441 is present only on the post-endoplasmic reticulum (ER)/cell surface pool of Kv1.2 and is not detectable on newly synthesized and ER-localized Kv1.2, on which we did observe pS449 phosphorylation. Elimination of PS440/PS441 phosphorylation by mutation reduces cell-surface expression efficiency and functional expression of homomeric Kv1.2 channels. Interestingly, mutation of S449 reduces phosphorylation at pS440/pS441 and also decreases Kv1.2 cell-surface expression efficiency and functional expression. These mutations also suppress trafficking of Kv1.2/Kv1.4 heteromeric channels, suggesting that incorporation of Kv1.2 into heteromeric complexes confers conditional phosphorylation-dependent trafficking to diverse Kv channel complexes. These data support Kv1.2 phosphorylation at these clustered C-terminal sites as playing an important role in regulating trafficking of Kv1.2-containing Kv channels.

Fig. 1.

Kv1.2 phosphorylation in mammalian brain and heterologous cells. (A) RBM and lysates from cells expressing Kv1.2 alone or Kv1.2 plus Kvβ2 were digested without (−) or with (+) AP, subjected to SDS/PAGE, and immunoblotted using a general Kv1.2-specific mAb (K14/16). Numbers on the left refer to molecular weight of prestained molecular weight standards. (B) Identification of phosphorylation sites on Kv1.2 in rat brain using LC-MS/MS. A doubly charged, doubly phosphorylated peptide at m/z 573.035, derived from Kv1.2 purified from rat brain, was fragmented to produce this MS/MS with y- and b-ion series that described the sequence IPpSpSPDLKK (amino acids 438–446). The phosphorylation sites were unambiguously assigned to S440 and S441 because of mass assignments from β-eliminated y6, y7, b3, and b4 fragment ions with neutral loss of phosphoric acid H₃PO₄ and 2H₃PO₄.

Fig. 2.

Relative SILAC quantification of phosphorylated peptides between ER and cell-surface forms of Kv1.2. (A) A doubly charged peptide representing doubly phosphorylated Kv1.2 at pS440/pS441 (IPpSpSPDLKK) was abundant in the sample from the cell-surface Kv1.2 pool (Light, peak at m/z 572.76) but was not detected in the samples from the ER Kv1.2 pool (Heavy, lack of peak at m/z 580.76). (B) The corresponding triply charged unphosphorylated peptide (IPSSPDLKK) was found with similar mass peak intensities in both the cell surface (Light, peak at m/z 328.86) and ER (Heavy, peak at 334.21) Kv1.2 pools. (C) A representative doubly charged nonphosphopeptide (TLAQFPETLLGDPK) from Kv1.2 demonstrates the overall ratio (≈2:1) of Kv1.2 between cell surface (Light, peak at m/z 765.42) and ER (Heavy, peak at m/z 769.43) samples applied for SILAC analysis.

Fig. 3.

Phosphospecific Abs Kv1.2P and L64/4 specifically recognize cell-surface Kv1.2. (A) Immunoblot analysis performed against RBM prepared from postnatal day 15 (P15) brain and lysates from HEK293 cells expressing WT Kv1.2 and phosphorylation site mutants S440A/S441A, S440A, S441A, S449A, and S434A. Shown is immunoreactivity using the general anti-Kv1.2 mAb K14/16 (Upper) and using the pS440/pS441-specific Kv1.2P rabbit Ab (Lower). (B) Input into and products of immunoprecipitation reactions performed with Kv1.2P Ab on lysates from HEK293 cells expressing WT Kv1.2, S440A/S441A, and S449A and blotted with K14/16. (C) (Left) Input into and products of immunoprecipitation reactions performed with Kv1.2P and Kv1.2C Abs on lysates from HEK293 cells expressing WT Kv1.2, and blotted with K14/16. (Center) Input into and products of immunoprecipitation reactions performed with Kv1.2P and Kv1.2C Abs on lysates of RBM as assessed by immunoblotting with K14/16 Ab. (Right) Cell surface biotinylated (Surface) and intracellular nonbiotinylated (Unbound) pools of WT Kv1.2 in HEK293 cells were isolated by surface biotinylation and streptavidin–agarose enrichment/depletion, respectively, from a total biotinylated cell lysate (Total). Fractions were immunoblotted for total Kv1.2 with K14/16 (Upper) and with Kv1.2P (Lower) Abs. Numbers on the left in all immunoblots refer to the molecular weight of prestained molecular weight standards. (D) COS-1 cells transfected with WT Kv1.2 were double immunofluorescence stained with K1.2P (red) and K14/16 (green) after permeabilization (Top), with Kv1.2e (green) before and K14/16 (red) after permeabilization (Middle), or Kv1.2e (green) before and L64/4 (red) after permeabilization (Bottom).

Fig. 4.

Mutations at in vivo phosphorylation sites suppress Kv1.2 surface expression and Kv1.2 currents. (A) COS-1 cells transfected with WT Kv1.2, S440A/S441A, or S449A were double immunofluorescence stained with Kv1.2e before and K14/16 after permeabilization, and a SEI determined the percentage of Kv1.2-expressing (K14/16-positive) cells with Kv1.2e surface staining. Statistical significance was determined by one-way ANOVA followed by Turkey's post hoc test, and statistical significance was considered at *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (B) Whole-cell patch–clamp recordings from HEK293 cells expressing WT Kv1.2 (squares), S440A/S441A (circles), or S449A (triangles). The cells were held at −80 mV and step depolarized to +60 mV for 200 ms in +10-mV increments. Peak current amplitudes at each test potential were divided by the cell capacitance to obtain the current densities. Mean ± SE of current densities obtained (Kv1.2, n = 11; Kv1.2 S440A/S441A, n = 6; S449A, n = 4) were plotted against each test potential. (C) Dose-dependent effects on Kv1.4 surface expression in the presence of increasing amounts of WT Kv1.2, S440A/S441A, or S449A cDNA in COS-1 cells (n = three samples of 100 cells each). Symbols are the same as in B.