Interdomain cytoplasmic interactions govern the intracellular trafficking, gating, and modulation of the Kv2.1 channel

Abstract

Voltage-gated potassium (Kv) channels comprise four transmembrane alpha subunits, often associated with cytoplasmic beta subunits that impact channel expression and function. Here, we show that cell surface expression, voltage-dependent activation gating, and phosphorylation-dependent modulation of Kv2.1 are regulated by cytoplasmic N/C interaction within the alpha subunit. Kv2.1 surface expression is greatly reduced by C-terminal truncation. Tailless Kv2.1 channels exhibit altered voltage-dependent gating properties and lack the bulk of the phosphorylation-dependent modulation of channel gating. Remarkably, the soluble C terminus of Kv2.1 associates with tailless channels and rescues their expression, function, and phosphorylation-dependent modulation. Soluble N and C termini of Kv2.1 can also interact directly. We also show that the N/C-terminal interaction in Kv2.1 is governed by a 34 aa motif in the juxtamembrane cytoplasmic C terminus, and a 17 aa motif located in the N terminus at a position equivalent to the beta subunit binding site in other Kv channels. Deletion of either motif disrupts N/C-terminal interaction and surface expression, function, and phosphorylation-dependent modulation of Kv2.1 channels. These findings provide novel insights into intrinsic mechanisms for the regulation of Kv2.1 trafficking, gating, and phosphorylation-dependent modulation through cytoplasmic N/C-terminal interaction, which resembles alpha/beta subunit interaction in other Kv channels.

Figure 1.

The soluble cytoplasmic C terminus of Kv2.1 rescues surface expression of a tailless Kv2.1 mutant in COS-1 cells. Immunofluorescence staining of Kv2.1-WT and the ΔC416 truncation mutant coexpressed without or with soluble Kv2.1C or Kv4.3C polypeptides in COS-1 cells. A, B, Surface (red) versus total (green) staining for Kv2.1-WT (A) and ΔC416 (B). Note no detectable surface expression of the ΔC416 truncation mutant. C, D, Total immunostaining (green) for the soluble C terminus of Kv2.1 (2.1C; C) and Kv4.3C (D) showing even distribution throughout the cytoplasm. E, Surface (red) versus total (green) immunostaining of ΔC416 coexpressed with 2.1C, showing surface expression of ΔC416 (compare with B) colocalizing with relocalized 2.1C. F, The cytoplasmic C terminus of Kv4.3 (4.3C) failed to rescue surface expression of the tailless Kv2.1 mutant. F, Surface (red) versus total (green; top row) and total immunostaining (bottom row) of cells coexpressing ΔC416 (red) and 4.3C (green), showing no apparent rescue of ΔC416 surface expression and no colocalization of ΔC416 with 4.3C. Scale bars, 5 μm.

Figure 2.

Coexpression of Kv2.1-ΔC416 and 2.1C in HEK293 cells enhances surface expression of functional channels and restoration of WT voltage-dependent gating properties. A, Immunostaining for ΔC416 (red) and 2.1C (green) in HEK293 cells. The images (top) are extended focus images of 40 cross-sectional X–Z sections (0.35 μm) through the cell. The bottom panel below each cell is the cross-sectional view at the level of drawn line. Note the redistribution of both ΔC416 and 2.1C, which are now colocalized on the cell surface when coexpressed, as opposed to perinuclear and scattered cytoplasmic localization of ΔC416 and 2.1C, respectively, when expressed alone. Scale bar, 5 μm. B, Representative whole-cell current traces obtained from HEK293 cells of comparable cell capacitance, transfected with either Kv2.1-WT, ΔC416, or ΔC416 plus 2.1C (1:1). The cells were held at −100 mV and depolarized for 200 ms to +80 mV in 10 mV increments. Inset, Magnified ΔC416 current. C, Current density plot of whole-cell current recordings of 2.1-WT or mutant constructs (as in B). Data are presented as mean picoamperes per picofarad ± SEM (n = 7 each). D, E, Coexpression of ΔC416 with 2.1C in HEK293 cells leads to the restoration of WT voltage-dependent activation gating (D) but yields a further shift in steady-state inactivation gating (E) properties. Intracellular dialysis of AP (100 U/ml) led to similar magnitude of hyperpolarizing shifts in the half-maximal conductance (G_1/2) and half-maximal steady-state inactivation (Vi_1/2) properties of reconstituted (ΔC416 plus 2.1C) channels and WT-Kv2.1, but not ΔC416. Voltage-dependent activation (D) and steady-state inactivation (E) curves were generated as described in Materials and Methods, and the voltage-dependent parameters are detailed in Table 1.

Figure 3.

The soluble 2.1C fragment rescues the surface expression and voltage-dependent gating properties of delayed rectifier Kv currents from the tailless mutant in cultured rat hippocampal neurons. A, Immunofluorescence staining of transfected neurons. Top row, Neurons expressing either HA-ΔC416 or 2.1C-myc alone showing total staining for HA-ΔC416 (red) and the dendritic marker MAP2 (green) or 2.1C-myc (green), as indicated. Middle row, Neurons cotransfected with HA-ΔC416 and 2.1C-myc showing HA-ΔC416 surface staining (red) and 2.1C-myc total staining (green). Note the redistribution of HA-ΔC416 when coexpressed with 2.1C-myc. Bottom row, Neurons transfected with HA-Kv2.1-WT showing surface (red) and total (green) staining. Scale bars, 10 μm. B, Representative whole-cell I_K current recordings from neurons without or with the transfection of the above-mentioned Kv2.1 constructs as per the voltage-pulse protocol described in Materials and Methods. C, Current density plot of whole-cell I_K current recordings from the transfected or untransfected neurons as mentioned in B. Data are presented as mean picoamperes/picofarad ± SEM (n = 4 each). D, E, Coexpression of HA-ΔC416 and 2.1C-myc leads to the restoration of WT voltage-dependent activation gating (D) and steady-state inactivation gating (E) properties of neuronal I_K currents. A magnified view of the area marked with a rectangle on the G–V relationship curve is shown as an inset in D. The voltage-dependent activation and steady-state inactivation curves for I_K were generated as detailed in Materials and Methods, and the voltage-dependent parameters are detailed in Table 2.

Figure 4.

The cytoplasmic N terminus of the Kv2.1-ΔC416 truncation mutant interacts directly with 2.1C. A, B, Co-IP of 2.1C and ΔC416 from HEK293 cells cotransfected with ΔC416 and 2.1C (at 1:1 ratio), using KC (recognizing Kv2.1-WT and 2.1C but not ΔC416) (A) or Kv2.1e (recognizing Kv2.1-WT and ΔC416 but not 2.1C) (B). C, Co-IP of 2.1C with ΔC416-Chimera (S4–S5 linker of Kv2.1 replaced with that of Kv1.2) or 2.1N from HEK293 cells expressing either construct alone or together (at 1:1 ratio), using anti-Kv2.1N antibody (recognizing Kv2.1-WT, ΔC416-Chimera, and 2.1N but not 2.1C). Immunoprecipitation products were size-fractionated by 7.5% (A, B) or 9% (C) SDS-PAGE and analyzed by immunoblot/ECL with mAbs K39/25 (recognizing Kv2.1-WT and ΔC416 but not 2.1C) (A) or K89/41 (recognizing Kv2.1-WT and 2.1C but not 2.1N and ΔC416) (B, C). Numbers to the left of each panel refer to mobility of prestained molecular weight standards in kilodaltons. D, Representative nondenaturing sucrose density gradient sedimentation patterns of Kv2.1-WT and mutant constructs expressed in HEK293 cells, as indicated.

Figure 5.

Truncation analyses of determinants of Kv2.1 cell surface expression. A, Total immunofluorescence staining for ΔN16, ΔN40, ΔC376, and ΔC396 truncation mutants of Kv2.1 expressed in HEK293 cells, showing plasma membrane-associated localization of ΔN16 and ΔC376 mutants and RER-like localization of ΔN40 and ΔC396 mutants. Scale bar, 10 μm. B, Schematic diagram of the Kv2.1 C- and N-terminal truncation and internal deletion mutants used to define the minimal motifs required for efficient plasma membrane localization. In the right column, (+) indicates efficient cell surface localization, whereas (−) indicates no detectable surface expression, as determined by surface and total immunofluorescence staining assay.

Figure 6.

The proximal 67 aa residues in the cytoplasmic C terminus are essential for direct interaction of 2.1C with Kv2.1 N terminus. A, Lysates from HEK293 cells expressing Kv2.1-WT or 2.1N, soluble 2.1C, and 2.1C lacking the first 67 aa residues (2.1C_478–853), either alone or in combination, were subjected to immunoprecipitation with the Kv2.1N antibody (recognizes Kv2.1-WT, ΔC416, and 2.1N but not 2.1C). Immunoprecipitation products were size-fractionated by 9% SDS-PAGE and analyzed by immunoblot/ECL using the mAb K89/41 (recognizes Kv2.1-WT, 2.1C, and 2.1C_478–853 but not ΔC416 and 2.1N). Note that ΔC416 and 2.1N interact with 2.1C but not 2.1C_478–853. B, 2.1C interacts with the cytoplasmic N terminus of the channel protein comprising the full-length tetramerization (T1) domain. Lysates from HEK293 cells expressing Kv2.1-WT or 2.1N, 2.1N_1–139, 2.1C, and 2.1C_478–853, either alone or in combination, were subjected to immunoprecipitation with Kv2.1N antibody (recognizes Kv2.1-WT, 2.1N, and 2.1N_1–139 but not 2.1C). Immunoprecipitation products were size-fractionated by 9% SDS-PAGE and analyzed by immunoblot/ECL using mouse mAb K89/41 that recognizes Kv2.1-WT, 2.1C and 2.1C_478–853 but not 2.1N or 2.1N_1–139. Numbers to the left of each panel refer to mobility of molecular weight standards in kilodaltons.

Figure 7.

A Kv2-specific motif in the N-terminal T1 domain regulates interaction with the C terminus and surface expression of Kv2.1. A, Sequence alignment of a portion of the T1 domains of rat Kv1.2 (accession number AAA19867.1), Kv3.1 (accession number NP_036988.1), Kv4.2 accession number NP_113918), Kv2.1 (accession number NP_037318.1), and Kv2.2 (accession number EDM11519.1) α subunits. The amino acids highlighted with gray boxes indicate identity with Kv2.1, and blue are amino acids forming the Kv2 family-specific C-terminal interaction motif. Red are amino acids crucial for binding to cytoplasmic auxiliary subunits in Kv1 and Kv4 α subunits. B, Comparative structural models of the T1 domains of Kv1.2, Kv3.1, Kv4.2 (taken from published crystal structures deposited in Protein Data Bank), and Kv2.1 (predicted by homology modeling with the Kv3.1 T1 domain). The gray circle represents the conserved zinc binding motif in the Kv2, Kv3, and Kv4 T1 domains, and the asterisk (*) denotes the Kv2 family-specific loop. C, Immunofluorescence staining of COS-1 cells expressing Kv2.1-Δ55–71. Note the RER-like localization. Scale bar, 5 μm. D, Sucrose density gradient sedimentation patterns of Kv2.1-WT (native and heat-denatured) and the Kv2.1-Δ55–71 mutant. E, Lysates from HEK293 cells expressing Kv2.1-WT or 2.1N, 2.1N-Δ55–71, and 2.1C, either alone or in combination, were subjected to immunoprecipitation with the Kv2.1N antibody (recognizes Kv2.1-WT, 2.1N-Δ55–71, but not 2.1C). Immunoprecipitation products were size-fractionated by 9% SDS-PAGE and analyzed by immunoblot/ECL using mAb K89/41, which recognizes Kv2.1-WT and 2.1C but not 2.1N or 2.1N-Δ55–71. Numbers to the left of each panel refer to mobility of prestained molecular weight standards in kilodaltons.

Figure 8.

N/C interaction motifs regulate the phosphorylation-dependent modulation of Kv2.1 voltage-dependent gating. A, The Kv2.1-Δ55–71 mutant is constitutively phosphorylated. HEK293 cell lysates from cells expressing Kv2.1-WT or Kv2.1-Δ55–71 were digested without or with AP (100 U/ml for 2 h at 37°C) and subjected to immunoblot with anti-Kv2.1 antibody KC and phosphospecific anti-Kv2.1 antibodies S453P, S563P, S603P, and S715P, as detailed in Materials and Methods. Numbers to the left of each panel refer to mobility of prestained molecular weight standards in kilodaltons. Note that Kv2.1-Δ55–71 is constitutively phosphorylated at all four major phosphorylation sites. B, Current density plot of whole-cell patch-clamp recordings of Kv2.1-WT and Kv2.1-Δ55–71. Data are presented as mean picoamperes per picofarad ± SEM (n = 5 each). C, D, Phosphorylation-dependent modulation of the voltage-dependent gating properties of Kv2.1 is abolished in the Δ55–71 mutant. Voltage-dependent activation (C) and steady-state inactivation (D) curves for Δ55–71 with or without intracellular AP dialysis. Note that Δ55–71 behaves quite similar to the Kv2.1 tailless mutant ΔC416 (Fig. 2C) in that it exhibits a relatively hyperpolarized G–V curve and lack of robust response to AP dialysis relative to Kv2.1-WT (gray lines: −AP, dashed; +AP, dotted).