Dynamic modulation of the kv2.1 channel by SRC-dependent tyrosine phosphorylation

Abstract

The voltage-gated K(+) channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here, we show that tyrosine phosphorylation by Src plays a fundamental role in regulating Kv2.1-mediated K(+) current enhancement. We found that the level of expression of the Kv2.1 protein is increased by Src kinase. Using mass spectrometric proteomic techniques, we identified two novel phosphotyrosine sites, Y686 and Y810, in the cytoplasmic domains of Kv2.1. We found that Src-dependent phosphorylation at these sites affects Kv2.1 through distinct regulatory mechanisms. Whereas phosphorylation at Y686 regulates Kv2.1 activity similarly to the known site Y124, phosphorylation at Y810 plays a significant role in regulating the intracellular trafficking of Kv2.1 channels. Our results show that these two novel tyrosine phosphorylation sites of Kv2.1 are crucial to regulating diverse aspects of Kv2.1 channel function and provide novel insights into molecular mechanisms for the regulation of Src-dependent modulation of Kv2.1 channels.

Fig. 1

Tyrosine phosphorylation of the Kv2.1 channel is induced by pervanadate treatment. (A) After ionomycin, carbachol, or pervanadate treatment, tyrosine phosphorylation of Kv2.1 was detected only in the pervanadate-treated sample. HEK293 cells were transfected with Kv2.1 expression plasmid; cells were treated with DMSO, ionomycin, carbachol, or pervanadate, lysed and immunoprecipitated with anti-Kv2.1 mAb. The immunoprecipitation products were separated by 7.5% SDS-PAGE and immunoblotted with anti-Kv2.1 and anti-PY-20 mAbs. (B) The effect of pervanadate concentration on Kv2.1 tyrosine phosphorylation. HEK293 cells were transfected with Kv2.1 expression plasmid. Cells treated with pervanadate (0, 25, 100, or 300 μM) were lysed and immunoprecipitated with anti-Kv2.1 (top and middle) and anti-PY20 (bottom). The immunoprecipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-PY20 (top) and anti-Kv2.1 mAbs (middle and bottom).

Fig. 2

Identification of Kv2.1 tyrosine phosphorylation sites Y686 and Y810. (A) Immunopurification of Kv2.1. Coomassie brilliant blue-stained SDS gel of a large-scale immunopurification showing the yield of Kv2.1 channel. (B) Cartoon of the membrane topology of Kv2.1 showing tyrosine phosphorylation sites identified by LC-MS/MS analyses. Black dots mark the novel tyrosine phosphorylation sites identified in this study; white dots mark tyrosine phosphorylation sites identified in a previous study. (C) MS/MS spectrum of Kv2.1 tyrosine phosphopeptide pY686. Shown is the product ion spectrum of a triply charged, singly phosphorylated tryptic peptide at m/z 982.20. (D) MS/MS spectrum of Kv2.1 tyrosine phosphopeptide pY810. Shown is the product ion spectrum of a triply charged, singly phosphorylated tryptic peptide at m/z 637.34.

Fig. 3

Src-induced increases in the expression level and activity of the Kv2.1 channel. (A) Kv2.1 protein levels are increased by Src-dependent tyrosine phosphorylation. Kv2.1-WT was co-transfected with Src variants (Src-DN, Src-CA and Src-WT) in HEK293 cells. Cells were lysed and immunoprecipitated using anti-pY20. The immunoprecipitation products were separated by 7.5% SDS-PAGE and immunoblotted with anti-Kv2.1 mAb. Short, short exposure; Long, long exposure. (B) Kv2.1-mediated K⁺ currents are increased by Src-dependent tyrosine phosphorylation. Whole cell patch recordings obtained from HEK293 cells co-expressing Kv2.1-WT with Src variants (Src-DN, Src-CA and Src-WT) are shown. Data are from HEK293 cells. (C) Mean ± S.E.M. current densities from HEK293 cells co-expressing Kv2.1-WT and Src variants (Src-DN (n=7), Src-CA (n=10), and Src-WT (n=10) or pcDNA (n=8)). Data are from HEK293 cells, asterisks represent samples that are significantly different (p< 0.05) from pcDNA.

Fig. 4

The Y686 and Y810 residues of the Kv2.1 channel are phosphorylated by Src. (A) PV phosphorylates the Y124, Y686, and Y810 residues of Kv2.1 via Src kinase. HEK293 cells were transfected with Kv2.1-WT or mutants (Y124F, Y686F, and Y810F). Cells were pretreated with or without Src kinase inhibitor PP2 before PV treatments. Cells were lysed and immunoprecipitated with anti-pY20 mAb. The immunoprecipitation products were separated by 7.5% SDS-PAGE and immunoblotted with anti-Kv2.1 mAb. Short, short exposure; Long, long exposure. (B) Mutating Y124, Y686 and Y810 decreases tyrosine phosphorylation of Kv2.1. HEK293 cells were co-transfected with Kv2.1-WT or mutants (Y124F, Y686F, and Y810F) and Src-CA. Cells were lysed and immunoprecipitated with anti-pY20 mAb. The immunoprecipitation products were separated by 7.5% SDS-PAGE and immunoblotted with anti-Kv2.1 mAb. (C) Kv2.1 tyrosine phosphorylation levels, normalized to Kv2.1 protein expression, are shown. Data are expressed as the mean ± S.E.M. (n=3, * P < 0.05, ** P < 0.01, Student's t test).

Fig. 5

Effects of the constitutively active form of Src, Src-CA, on outward K⁺ currents in Kv2.1-expressing HEK cells using the whole-cell patch clamp technique. (A) (Left) Current-voltage relations of WT Kv2.1 channels expressed in the absence (n = 14, filled symbols) or presence of Src-CA (n = 15, open symbols). (Right) Representative whole-cell K⁺ currents and mean ± S.E.M. current densities from HEK cells expressing Kv2.1-WT or Kv2.1-WT and Src-CA (B) (Left) Current-voltage relations of Kv2.1-Y124F channels expressed in the absence (n = 7, filled symbols) or presence of Src-CA (n = 13, open symbols). (Right) Representative whole-cell K⁺ currents and mean (± S.E.M.) current densities in HEK cells expressing Kv2.1-Y124F or Kv2.1-Y124F and Src-CA. (C) (Left) Current-voltage relations of Y686F Kv2.1 channels expressed in the absence (n = 9, filled symbols) or presence of Src-CA (n = 12, open symbols). (Right) Representative whole-cell K⁺ currents and mean (± S.E.M.) current densities in HEK cells expressing Kv2.1-Y686F or Kv2.1-Y686F and Src-CA. (D) (Left) Current-voltage relations of Kv2.1-Y686F channels expressed in the absence (n = 20, filled symbols) or presence of Src-CA (n = 19, open symbols). (Right) Representative whole-cell K⁺ currents and mean (± S.E.M.) current densities in HEK cells expressing Kv2.1-Y686F or Kv2.1-Y686F and Src-CA. (*P < 0.01, **P < 0.05; Student's t-test). Current density was calculated as current amplitude at +30 mV (normalized to cell capacitance) of the K⁺ current evoked by sequential 10 mV voltage steps to +50 mV from a holding potential of −60 mV. Calibration: 300 pA, 100 ms.

Fig. 6

Tyrosine phosphorylation of the Kv2.1 channel at Y810 is critical for the membrane trafficking of the channel by Src. (A) HEK293 cells were cotransfected with Kv2.1-WT or mutants (Y124F, Y686F, and Y810F) and Src-CA. Cells were then incubated with sulfo-NHS-SS-biotin, and biotinylated proteins were analyzed by immunoblotting with anti-Kv2.1. Eluates from avidin beads and total lysates were separated by 7.5% SDS-PAGE and compared. (B) The intensity of surface Kv2.1 levels, normalized to total Kv2.1 protein expression, is shown. Data are expressed as the mean ± S.E.M. (n=4, * P < 0.05, ** P < 0.01, Student's t test).