The Electrically Silent Kv5.1 Subunit Forms Heteromeric Kv2 Channels in Cortical Neurons and Confers Distinct Functional Properties

Abstract

Voltage-gated K⁺ channels of the Kv2 family are highly expressed in brain and play dual roles in regulating neuronal excitability and in organizing endoplasmic reticulum-plasma membrane (ER-PM) junctions. Studies in heterologous cells suggest that Kv2.1 and Kv2.2 co-assemble with "electrically silent" KvS subunits to form heterotetrameric channels with distinct biophysical properties, but the prevalence and localization of these channels in native neurons are unknown. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain of both sexes, the most abundant being Kv5.1. We found that Kv5.1 co-immunoprecipitates with Kv2.1 and to a lesser extent with Kv2.2 from brain lysates and that Kv5.1 protein levels are decreased by 70% in Kv2.1 knock-out mice and 95% in Kv2.1/Kv2.2 double knock-out mice. RNAscope and immunolabeling revealed that Kv5.1 is prominently expressed in neocortex, where it is detected in a substantial fraction of Kv2.1/Kv2.2 positive neurons in layers 2/3, 5, and 6. At the subcellular level, Kv5.1 protein is coclustered with Kv2.1 and Kv2.2 at presumptive ER-PM junctions on the soma and proximal dendrites of cortical neurons. Moreover, in addition to modifying channel conductance, we found that Kv2/Kv5.1 channels are less phosphorylated and insensitive to RY785, a potent and selective Kv2 channel inhibitor. Together, these findings demonstrate that KvS subunits create multiple Kv2 channel subtypes in brain. Most notably, Kv2/Kv5.1 channels are highly expressed in cortical neurons, where their unique properties likely modulate the critical conducting and nonconducting roles of Kv2 channels.

Keywords: ER-PM junction; channel localization; modulatory subunit; voltage-gated potassium channel.

Figure 1.

Kv5.1 co-assembles with Kv2 subunits to form heteromeric channels. A, Mass spectrometry-based proteomics analyses of Kv2.1 complexes immunopurified from cross-linked adult mouse brain samples. The identified proteins are listed by their mean spectral abundance, expressed as a percentage of Kv2.1 spectral counts (mean ± SEM, n = 3). Several KvS subunits copurified with Kv2 channels, with Kv5.1 being the most abundant. B, HEK cells expressing Kv2.1 plus Kv5.1 or other myc-tagged KvS subunits were solubilized, size fractionated on SDS gels and immunoblotted for Kv5.1 with polyclonal antibody 5.1C (top) or monoclonal antibody L134/44 (middle) and for myc and Kv2.1 (bottom). Both Kv5.1 antibodies detected Kv5.1 but no other KvS subunit proteins. Numbers to the left are molecular weights standards in kD. C, Kv5.1 was immunoprecipitated from rat brain lysates with 5.1C antibody and immunoblotted with polyclonal antibody 5.1C (left) or monoclonal antibody L134/44 (right). Both antibodies recognized only the characteristic Kv5.1 doublet bands at ∼52kD. D, HEK cells expressing GFP-tagged Kv5.1 and Kv2.1 or Kv2.2, as designated in the lane labels (c, untransfected cells), were solubilized in RIPA buffer and immunoprecipitations (IPs) performed using affinity-purified Kv5.1C antibody. The IP reactions were size fractionated on SDS gels and immunoblotted for GFP. Kv2.1 and Kv2.2 were both co-IPed with Kv5.1, consistent with their co-assembly into heteromeric channels. Arrows to the right denote positions of the target proteins. E, Live HEK cells expressing Kv2.1 and/or Kv5.1 with an extracellular bungarotoxin binding site (BBS) were cell surface labeled with AF647-Btx (blue), and then fixed, permeabilized, and immunolabeled with anti-Kv2.1 (magenta) and Kv5.1 (green) antibodies. Kv5.1BBS was detected on the cell surface with Btx and anti-Kv5.1 antibody only when coexpressed with Kv2.1. Scale bar, 10 μm. F, HEK cells expressing HA-Kv5.1BBS alone or HA-Kv2.1 + HA-Kv5.1BBS were either labeled live with biotin-Btx (surface) or extracted and then labeled with biotin-Btx (total). The streptavidin precipitation reactions were size fractionated on SDS gels and immunoblotted for the HA epitope tag. Kv5.1 was detected on the cell surface only when coexpressed with Kv2.1. DsRed-HA-Kv2.1BBS is shown as a positive control for surface expression. Arrows to the right denote positions of the IPed proteins.

Figure 2.

Kv5.1 is a common component of native Kv2 channels in brain. A, Rat brain membranes (RBM) were solubilized with RIPA buffer and IPs performed using polyclonal antibodies against Kv2.1, Kv1.2, or Kv5.1 (s, serum; p, affinity-purified). The RBM starting material and the reaction products from IPs performed with the various antibodies as designated in the lane labels were size fractionated on SDS gels and immunoblotted for Kv2.2 (green), and Kv2.1 (red). The panel below shows a section of the blot that was probed for Kv1.2. Both Kv2.1 and Kv2.2 were robustly co-IPed together with Kv5.1, but not with Kv1.2. Labels to the right denote positions of the target proteins. Numbers to the left are molecular weights standards in kD. B, Complementary IPs were performed from mouse brain lysates using Kv2.1 or Kv2.2 subunit antibodies. The input lysate (I), the post-IP depleted lysates (top panels), and the IP fractions (bottom panels) performed with the various antibodies as designated in the lane labels were size fractionated on SDS gels. Immunoblotting of pre- and post-IP lysates (top panel) for Kv2.2 (green) and Kv2.1 (red) confirmed that both IPs were highly efficient. The panel below shows a section of the blot that was probed for GRP75/Mortalin as a loading control. Immunoblotting of the IP fractions (bottom panel) for Kv5.1 shows that Kv5.1 co-IPed together with Kv2.1 and to a lesser extent with Kv2.2, but not with Kv1.2. Labels to the right denote positions of the target proteins. Numbers to the left are molecular weights standards in kD. Asterisk denotes the light chain of the rabbit antibodies used in the IPs. C, Top graph, Quantification of pre- and post-IP brain lysates showed that 87% of Kv2.2 and 79% of Kv2.1 protein were depleted from brain lysates with Kv2.2 and Kv2.1 IPs, respectively (n = 3–5). Bottom graph, Quantification of Kv2.2, Kv2.1 and Kv5.1 subunits in each IP, normalized to the amount IPed by each subunit-specific antibody (n = 4–6, one-way ANOVA and Sidak's multiple-comparisons test, ***p < 0.001, **p < 0.01, *p < 0.05). Approximately 21–22% of Kv2.1 and Kv2.2 were co-IPed with the other Kv2 subunit, and 16% of Kv2.1 and 7% of Kv2.2 were co-IPed together with Kv5.1. Twice as much Kv5.1 was co-IPed together with Kv2.1 as compared with Kv2.2. D, Mass spectrometry-based proteomics analyses of Kv2 channels immunopurified from non-cross-linked brain lysates using Kv2.1- and Kv5.1-specific antibodies. Mean spectral abundance is expressed as a percentage of Kv2.1 spectral counts (mean ± SEM, n = 3). Several KvS subunits were detected in Kv2.1 IPs, with Kv5.1 being the most abundant (18.6% of Kv2.1 levels). In contrast, no other KvS subunits were detected in Kv5.1 IPs.

Figure 3.

Kv5.1 protein is significantly reduced in Kv2 KO brain. A, Immunoblots of brain lysates from wild-type (WT), Kv2.1 KO, and Kv2.1/Kv2.2 DKO mice show that Kv5.1 protein levels are severely reduced in Kv2 KO brain. Labels to the right denote positions of the target proteins. Numbers to the left are molecular weights standards in kD. B, Immunoblots of Kv5.1 IPs from brain lysates of wild-type (WT), Kv2.1 KO, and Kv2.1/Kv2.2 DKO mice show that Kv5.1 protein levels are severely reduced in Kv2.1 KO and Kv2.1/Kv2.2 DKO brain. Asterisk denotes the light chain of the rabbit 5.1C antibody used in the IPs. Graph shows quantitation of IP reaction products. Compared with WT mouse brain, the amount of Kv5.1 IPed is decreased by 70% in Kv2.1 KO and 95% in Kv2.1/Kv2.2 DKO brain samples (one-sample t and Wilcoxon test, n = 4–5, **p = 0.0018, ****p < 0.0001). Thus, Kv5.1 expression is dependent on Kv2 subunits and primarily on Kv2.1. C, HEK cells transfected with Kv5.1 alone, Kv5.1 + Kv2.1, or Kv2.1 alone were solubilized, size fractionated on SDS gels, and immunoblotted with the designated antibodies. The amount of Kv5.1 protein was significantly higher (∼180%) in cells expressing Kv5.1 + Kv2.1 as compared with Kv5.1 alone (one-sample t and Wilcoxon test, n = 6, **p < 0.01). The amount of Kv5.1 plasmid was kept constant for both transfections. D, Kv5.1 and Kv2.1 were IPed from HEK cell lysates transfected as in C, and IBed with antibodies to Kv5.1 (left gel) and ubiquitin (P4D1, right gel). In addition to the major Kv5.1 band at 52kD, high molecular weight bands were detected for Kv5.1 that overlapped with bands for ubiquitin (bracket). Similar laddering was not evident for Kv2.1. The amount of Kv5.1 and ubiquitin laddering were lower in cells expressing Kv5.1 + Kv2.1 as compared with Kv5.1 alone, when normalized to the amount of Kv5.1 IPed (one-sample t and Wilcoxon test, n = 4–5, **p < 0.01).

Figure 4.

Kv5.1 protein is highly expressed in cortex. A, A sagittal section of mouse brain perfusion fixed with GAA and immunolabeled for Kv2.1 (magenta) and Kv5.1 (green). Kv5.1 immunolabeling is prominent in the neocortex and subiculum, where it overlaps substantially with Kv2.1 immunolabeling (white indicates magenta + green overlap). Abbreviations: CTX, cortex; Sub, subiculum; HC, hippocampus; ST, striatum; TH, thalamus; MB, midbrain; CB, cerebellum. Scale bar, 1,000 µm. B, Higher magnification images of Kv2.1/Kv5.1 immunofluorescent labeling in different brain regions. In the neocortex, Kv5.1 (green) immunolabeling is detected in a large fraction of neurons that express Kv2.1 (magenta) and their immunolabeling is extensively colocalized at the cellular level (arrow). Robust Kv5.1 immunolabeling that colocalizes with Kv2.1 is also detected in many neurons in subiculum (arrow) but is lacking in adjacent CA1 pyramidal neurons in hippocampus. In most other brain regions, cellular Kv5.1 immunolabeling is weak [e.g., thalamus (arrow)] or undetectable (e.g., striatum). Some diffuse Kv5.1 labeling is also apparent throughout neuropil in thalamus that does not correspond to cellular Kv2.1 labeling (arrowhead). Images were acquired with the same exposure times and were subjected to identical linear adjustments of min/max signals for display purposes. Scale bar, 20 µm.

Figure 5.

Kv5.1 protein is differentially expressed across cortical layers. A, Multiplex immunofluorescent labeling of a sagittal section of mouse somatosensory cortex. Kv5.1 (green) immunolabeling varies across cortical layers and is most prominent in layers 2/3, 5 and 6, where it is detected in a significant subpopulation of neurons that express Kv2.1 (blue) and/or Kv2.2 (magenta). Kv5.1 immunolabeling is low in L4. Scale bar, 50 µm. B, Line scan (of 100 µm band) shows the relative immunolabeling intensity for Kv2.1, Kv2.2, and Kv5.1 across cortical layers. C, Higher magnification images showing that Kv5.1 immunolabeling is restricted to neurons expressing Kv2.1 and/or Kv2.2, depending on the cortical layer. Scale bar, 10 µm.

Figure 6.

Kv5.1 transcript is differentially expressed across cortical layers. A, Coronal cryostat sections of mouse cortex were hybridized using RNAscope with probes targeting Kcnf1 (Kv5.1) and then immunolabeled for Kv2.1. Prominent labeling for Kv5.1 transcript was evident in cortical layers 2/3, 5, and 6 and was confined to neurons that immunolabeled for Kv2.1 protein. Scale bar, 50 µm. B, Mean intensity of Kcnf1 RNAscope probe labeling in Kv2.1-positive neurons in each cortical layer (n = 30–43 neurons per layer). In layers 2/3, 5, and 6, a large fraction of Kv2.1-positive neurons exhibited Kcnf1 probe labeling and the intensity of probe labeling varied between neurons across a broad range. The dashed line (at 2× mean background labeling) represents the approximate division between Kv5.1-positive and Kv5.1-negative neurons.

Figure 7.

Kv5.1 protein is differentially expressed in L2/3 neurons and colocalizes with Kv2 subunits. A, Confocal image of multiplex immunofluorescent labeling of L2/3 neurons shows that Kv5.1 (green) colocalizes extensively with both Kv2.1 (blue) and Kv2.2 (magenta) on somata and proximal dendrites. However, the ratios of Kv2.1, Kv2.2, and Kv5.1 immunolabeling vary widely between neighboring neurons (note varying hues of immunolabeling). Similar Kv5.1 immunolabeling was obtained with 5.1C polyclonal (top) and L134/44 monoclonal (bottom) antibodies. Scale bar, 10 µm. B, In L2/3 neurons, Pearson's correlation coefficient (PCC) values are similar for Kv2.1 versus Kv5.1 and Kv2.2 versus Kv5.1, indicating that Kv5.1 colocalizes with both Kv2 subunits (one-way ANOVA and Tukey's multiple-comparisons test, ****p < 0.0001, **p = 0.0026, n = 33 neurons, 3 WT brains). In contrast, PCC values for Kv2.1 or Kv5.1 versus GAD67-labeled inhibitory terminals on the neuronal soma are much lower, reflecting little colocalization (n = 13 neurons). C, In L2/3 neurons, simple linear regression shows that the mean intensity of Kv5.1 immunolabeling correlates with that of Kv2.1 (**p < 0.001, PCC = 0.41), and to a slightly lesser extent with Kv2.2 (p < 0.01, PCC = 0.33, n = 68 neurons). D, Top row, Combined RNAscope using probes for Kcnf1 (Kv5.1) transcript and Kv2.1 immunolabeling of L2/3 neurons. Kv5.1 transcript is detected at varying levels in most Kv2.1-positive neurons (∼84%). Bottom row, Combined RNAscope using probes for Kcnf1 (Kv5.1) transcript and Kv5.1 (L134/44) immunolabeling of L2/3 neurons. Kv5.1 mRNA labeling and protein immunolabeling are detected in the same neurons. Scale bar, 10 µm.

Figure 8.

Kv5.1 immunolabeling is significantly reduced in Kv2.1 KO brain. A, Immunolabeling of L2/3 neurons for Kv5.1 (green), Kv2.1 (blue), and Kv2.2 (magenta) in WT (top) and Kv2.1 KO (bottom) mouse brain sections. Immunolabeling shows that the intensity of Kv5.1 immunolabeling associated with the somatic PM is undetectable or severely reduced in L2/3 neurons in Kv2.1 KO brain as compared with wild type (WT). Images were acquired with the same exposure times and were subjected to identical linear adjustments of min/max signals for display purposes. Scale bar, 20 µm. B, The reduced levels of Kv5.1 immunolabeling persisting in certain Kv2.1 KO neurons colocalize with Kv2.2 immunolabeling. Intensity profile plots show that this residual Kv5.1 is coclustered with Kv2.2 on the plasma membrane. Scale bar, 10 µm. C, D, PCC values for Kv2.1, Kv2.2, and Kv5.1 immunolabeling in L2/3 neurons in WT and Kv2.1 KO brain. Using both polyclonal (C) and monoclonal (L134/44; D) Kv5.1 antibodies, the PCCs for Kv2.2 versus Kv5.1 immunolabeling are significantly reduced in Kv2.1 KO brain as compared with WT (one-way ANOVA and Sidak's multiple-comparisons test, ****p < 0.0001, n = 33 WT and 18 KO neurons for C, and 33 WT and 38 KO neurons for D, n = 3 pairs of WT and Kv2.1 KO brains). E, Mean intensity of Kv5.1 immunolabeling in Kv2.2 positive L2/3 neurons in a representative pair of WT and Kv2.1 KO brains. Kv5.1 immunolabeling is significantly reduced in L2/3 neurons in Kv2.1 KO as compared with WT brain (one-way ANOVA and Sidak's multiple-comparisons test, ****p < 0.0001, ***p < 0.001, points represent individual neurons).

Figure 9.

Kv5.1/Kv2 channels are clustered at ER-PM junctions in brain neurons. A, In cortical L2/3 neurons, immunolabeling shows that Kv5.1 (green) is coclustered with Kv2.1 (blue) and Kv2.2 (magenta) on neuronal soma at presumptive ER-PM junctions. This is evident in the nonuniformity and coincidence of the three labels in the intensity profile (B). Scale bar, 2.5 µm. C, Clustering of Kv2.1, Kv2.2 and Kv5.1 was compared in L2/3 neurons by measuring the coefficient of variation of labeling intensity (CV: SD/mean of pixel intensity). CV values were similar for Kv2.1, Kv2.2, and Kv5.1, suggesting that there is no significant difference in the clustering of Kv2- and Kv5.1-containing channels (one-way ANOVA and Tukey's multiple-comparisons test, p > 0.05, n = 59 neurons, 3 WT brains, black and orange points indicate immunolabeling experiments using Kv5.1 polyclonal and monoclonal antibodies, respectively). D, In L2/3 neurons, the CV values of Kv2.1 and Kv5.1 immunolabeling are correlated (Pearson's correlation coefficient r = 0.61, p < 0.001, n = 35 neurons) and unrelated to Kv5.1 immunofluorescent labeling intensity (5.1IF). E, In L2/3 neurons, the CV values for Kv2.1 and Kv2.2 immunolabeling are not correlated with Kv5.1 immunolabeling intensity (r = 0.04 and −0.26, n = 35 neurons). F, Immunolabeling shows that Kv5.1 clusters in L2/3 neurons (magenta) commonly overlap with or are juxtaposed to SPHKAP puncta (green), which localize to stacked ER cisternae at ER-PM junctions. Scale bar, 5 µm.

Figure 10.

Kv5.1 subunits alter Kv2 channel phosphorylation. A, Kv2.1 C-terminal domain phosphorylation was assessed by proteomic analysis of Kv2.1 protein immunopurified from brain samples with Kv2.1 or Kv5.1 antibodies. Left panel, The fraction of Kv2.1 spectra containing phosphorylated S/T residues was lower in Kv5.1-containing channels compared with total Kv2.1 channels (paired t test, *p = 0.03, n = 3). Right panel, Kv2.1 phosphorylation was decreased by ∼40% in Kv2/Kv5.1 heteromeric channels compared with all Kv2.1 channels (one-sample t test, *p = 0.03, n = 3). B, Kv5.1 C-terminal domain phosphorylation was assessed by proteomic analysis of Kv5.1 protein immunopurified from brain samples. Five S/T residues were phosphorylated to varying degrees. Notably, phosphorylation of either S470 or S472 was observed in a large fraction of those spectra (mean ± SEM = 0.82 ± 0.07), but phosphorylation of both sites was never observed.

Figure 11.

Kv5.1/Kv2.1 channels have distinct biophysical properties and pharmacology. A, Exemplar current traces from Kv2.1-CHO cells before (black) and after (red) application of 1 µM RY785. Left panel, Transfection control. Right panel, Kv2.1-CHO cells transfected with Kv5.1. B, Current remaining after application of 1 μM RY785 as in panel A. Black bars represent mean. Each point is current from one cell at the end of a 200 ms, 0 mV voltage step. Currents >100% remaining indicates current increase after addition of RY785. t test ***p < 0.001. C, Voltage dependence of activation normalized to maximum conductance of initial tail currents at 0 mV. Mean ± SEM. Kv2.1/control (black) n = 6 cells, Kv2.1/Kv5.1 (green) n = 7 cells. Lines are Boltzmann fits (Eq. 1) Kv2.1/control: V_1/2 = 2.6 ± 1 mV; z = 1.6 ± 0.1 e₀, Kv5.1/Kv2.1: V_1/2 = 15 ± 2 mV; z = 1.4 ± 0.1 e₀. D, Exemplar traces of channel deactivation at −40 mV after a 50 ms step to +20 mV. Traces are normalized to max current during −40 mV step. E, The faster time constant of double exponential fits to channel deactivation. t test p = 0.001.