Convergent modulation of Kv4.2 channel alpha subunits by structurally distinct DPPX and KChIP auxiliary subunits

Abstract

Kv4.2 is the major voltage-gated K(+) (Kv) channel alpha subunit responsible for the somatodendritic transient or A-type current I(SA) that activates at subthreshold membrane potentials. Stable association of Kv4.2 with diverse auxiliary subunits and reversible Kv4.2 phosphorylation regulate I(SA) function. Two classes of auxiliary subunits play distinct roles in modulating the biophysical properties of Kv4.2: dipeptidyl-peptidase-like type II transmembrane proteins typified by DPPX-S, and cytoplasmic Ca(2+) binding proteins known as K(+) channel interacting proteins (KChIPs). Here, we characterize the convergent roles that DPPX-S and KChIPs play as component subunits of Kv4.2 channel complexes. We coexpressed DPPX-S with Kv4.2 in heterologous cells and found a dramatic redistribution of Kv4.2, releasing it from intracellular retention and allowing plasma membrane expression, as well as altered Kv4.2 phosphorylation, detergent solubility, and stability. These changes are remarkably similar to those obtained upon coexpression of Kv4.2 with the structurally distinct KChIPs1-3 auxiliary subunits. KChIP4a, which negatively affects the impact of other KChIPs on Kv4.2, also inhibits the effects of DPPX-S, consistent with the formation of a ternary complex of Kv4.2, DPPX-S, and KChIPs early in channel biosynthesis. Tandem MS analyses reveal that coexpression with DPPX-S or KChIP2 leads to a pattern of Kv4.2 phosphorylation in heterologous cells similar to that observed in brain, but lacking in cells expressing Kv4.2 alone. In conclusion, transmembrane DPPX-S and cytoplasmic KChIPs, despite having distinct structures and binding sites on Kv4.2, exert similar effects on Kv4.2 trafficking, but distinct effects on Kv4.2 gating.

FIGURE 1

DPPX-S co-expression leads to changes in the subcellular localization of Kv4.2 expressed in COS-1 cells. (A–C) COS-1 cells were transfected with (A) Kv4.2 alone; or (B) Kv4.2 plus DPPX-S; and (C) Kv4.2 plus KChIP2 at 1:1 cDNA ratios, and stained for total and cell surface Kv4.2 to determine the extent of Kv4.2 cell surface expression in transfected cells. Total cellular Kv4.2 pool (left panels). Cell surface Kv4.2 pool (right panels). (D–E) COS-1 cells transfected with either (D) DPPX-S alone or (E) DPPX-S and Kv4.2. Cells were permeabilized and stained for DPPX-S. Scale bar = 20 μm. (F) Dose-response curve showing changes in surface expression index (SEI: the percentage of Kv4.2-expressing cells with Kv4.2 cells surface expression) in response to increasing amounts of co-transfected DPPX-S cDNA. Three independent dishes were assayed for each Kv4.2/DPPX-S ratio. Approximately 100 Kv4.2 expressing cells were counted from each sample. Data are presented as the mean +/− S.E.

FIGURE 2

DPPX-S induces a dose-dependent shift in the M_r of Kv4.2 on SDS gels. COS-1 cells were co-transfected with a fixed amount of Kv4.2 cDNA and increasing amounts of DPPXS. (A) Detergent extracts were immunoblotted for Kv4.2 and DPPX-S. (B) Quantitative analysis of the DPPX-S dose dependent effects on the electrophoretic mobility of Kv4.2. The intensities of the immunoreactivity of the M_r ≈ 65 kDa form (upward facing arrowheads and error bars) and M_r ≈ 70 kDa form (downward facing arrowhead and error bars) at each cDNA ratio were measured as the percentage of each form relative to the total Kv4.2 pool. (C) Crude membrane fractions from COS-1 cells expressing Kv4.2 alone, Kv4.2 + DPPX-S, or Kv4.2 + KChIP2 were incubated in the presence (+) or absence (−) of alkaline phosphatase (AP) and immunoblotted for Kv4.2.

FIGURE 3

DPPX-S-dependent phosphorylation of Kv4.2 at Serine 552. (A) Detergent extracts of COS-1 cells expressing Kv4.2 alone, Kv4.2 and KChIP2, and Kv4.2 and DPPX-S were immunoblotted with phospho-independent (K57/41, left panel) or phospho-specific (S552P) antibody (right panel). Note that the S552P antibody recognizes only the M_r ≈ 70 kDa form of Kv4.2. (B) Kv4.2 from COS-1 cell lysates expressing Kv4.2, Kv4.2 and KChIP2, and Kv4.2 and DPPX-S was subjected to immunoprecipitation using an N-terminal directed phosphoindependent antibody (Kv4.2N) or the phospho-specific S552P antibody. Samples were immunoblotted with anti-Kv4.2 (K57/41; upper panel) or S552P (lower panel) antibodies.

FIGURE 4

Phosphorylation at Serine 552 is specific to the cell surface Kv4.2 pool. (A–C) COS- 1 cells were transfected with Kv4.2 alone (A), Kv4.2 and DPPX-S (B), or with Kv4.2 and KChIP2 (C) at 1:1 cDNA ratios and stained for Kv4.2 using phospho-independent (K57/41, left panels) or S552P phosphospecific (right panels) antibodies. Scale bar = 20 μm.

FIGURE 5

Molecular characteristics of Kv4.2 are altered upon co-expression with DPPX-S. (A) COS-1 cells expressing Kv4.2 alone or Kv4.2 and DPPX-S at a 1:1 ratio were harvested and solubilized in lysis buffer with and without the non-ionic detergent Triton X-100 as indicated. Detergent insoluble (I) and soluble (S) cell fractions were separated by centrifugation and immunoblotted for Kv4.2 (K57/41). (B) Quantitative analysis of the detergent solubility of the DPPX-S dependent 70 kDa population of Kv4.2 with 1% Triton X-100. The intensities of the immunoreactivity of the M_r ≈ 65 kDa form (black columns and error bars) and M_r ≈ 70 kDa form (white columns and error bars) for the insoluble and soluble fractions of Kv4.2 were measured as a percentage of each form relative to the total pool. (C) Immunoblot analysis of the half-life of Kv4.2 in the absence and presence of DPPX-S. COS-1 cells expressing Kv4.2 alone or Kv4.2 and DPPX-S were incubated in the presence of cycloheximide (100 μg/ml) for the indicated time and immunoblotted for Kv4.2.

FIGURE 6

KChIP4a co-expression inhibits the DPPX-S induced effects on Kv4.2. (A) COS-1 cells were triple-transfected with Kv4.2 and DPPX-S at a 2:1 ratio (1 and 0.5 μg) and increasing amounts (0–2 μg) of KChIP4a cDNA was added as indicated. Detergent extracts of transfected cells were immunoblotted with anti-Kv4.2 and anti-DPPX antibodies. (B) Quantitative analysis of the KChIP4a inhibition of DPPX-S dependent effects on the electrophoretic mobility of Kv4.2. The intensities of the immunoreactivity of the M_r ≈ 65 kDa form (up facing arrowheads and error bars) and M_r ≈ 70 kDa form (squares and down facing error bars) at each cDNA ratio were measured as the percentage of each form relative to the total Kv4.2 pool. (C) Triple label immunofluorescence staining of COS-1 cells triple-transfected with Kv4.2, DPPX-S, and KChIP4a at 2:1:1 cDNA ratios. (D–E) COS-1 cells transiently transfected with KChIP4a (D) or DPPX-S (E) cDNA were stained for KChIP4a (K55/82) or DPPX (anti-HA). (F) COS-1 cells co-transfected with KChIP4a and DPPX-S cDNA at a 1:1 ratio were double-stained for KChIP4a and DPPX-S. Scale bar = 20 μm.

FIGURE 7

Identification of Kv4.2 phosphorylation sites by tandem mass spectrometry. MS/MS spectrum of Kv4.2 phosphopeptide GpSVQELSTIQIR from COS-1 cells co-expressing Kv4.2 and DPPX-S.

FIGURE 8

Effects of phosphorylation site point mutations. (A) Effects of phosphorylation site mutations on the M_r of Kv4.2 on SDS PAGE. Solubilized Kv4.2 from detergent extracts of COS- 1 cells expressing wild-type Kv4.2 or phosphorylation site mutants S548A, S552A, S572A, and S575A alone or with DPPX-S were size fractionated on SDS PAGE and immunoblotted for Kv4.2. (B) Effects of phosphorylation site mutations on the localization of Kv4.2 in transfected cells. Kv4.2 expressed with empty vector RBG4 (left panels) or with DPPX-S (right panels) were double-stained for anti-Kv4.2 and S552P as indicated. Scale bar = 20 μm.