Electrically silent KvS subunits associate with native Kv2 channels in brain and impact diverse properties of channel function

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 the two pore-forming alpha subunits Kv2.1 and Kv2.2 assemble with "electrically silent" KvS subunits to form heterotetrameric channels with distinct biophysical properties. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain, 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 knockout mice and 95% in Kv2.1/2.2 double knockout mice. Multiplex immunofluorescent labelling of rodent brain sections revealed that in neocortex Kv5.1 immunolabeling is apparent in a large percentage of Kv2.1 and Kv2.2-positive layer 2/3 neurons, and in a smaller percentage of layer 5 and 6 neurons. At the subcellular level, Kv5.1 is co-clustered with Kv2.1 and Kv2.2 at ER-PM junctions in cortical neurons, although clustering of Kv5.1-containing channels is reduced relative to homomeric Kv2 channels. We also found that in heterologous cells coexpression with Kv5.1 reduces the clustering and alters the pharmacological properties of Kv2.1 channels. Together, these findings demonstrate that the Kv5.1 electrically silent subunit is a component of a substantial fraction of native brain Kv2 channels, and that its incorporation into heteromeric channels can impact diverse aspects of Kv2 channel function.

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 crosslinked 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 co-purified with Kv2 channels, with Kv5.1 being the most abundant. (B) 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 an affinity-purified Kv5.1 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. Numbers to the left are molecular weights standards in kD. (C) 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 permeabilized and immunolabeled with anti-Kv2.1 (red) and Kv5.1 (green) antibodies. Kv5.1BBS was detected on the cell surface with Btx and anti-Kv5.1 antibody only when co-expressed with Kv2.1. Scale bar = 10 μm. (D) 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 co-expressed 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. Numbers to the left are molecular weights standards in kD. (E) Exemplar current traces from Kv2.1-CHO cells before (black) and after (green) application of 1 μM RY785. Left panel: transfection control. Right panel: Kv2.1-CHO cells transfected with Kv5.1. (F) 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. T test *** p<0.001. (G) 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 (Equation 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. (H) 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. (I) The faster time constant of double exponential fits to channel deactivation. T test p = 0.001.

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 to 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 panel) 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 it 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. (C) 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 (top graph; n=3-5). Quantification of Kv2.2, Kv2.1 and Kv5.1 subunits in each IP, normalized to the amount IPed by each subunit-specific antibody (bottom graph; n=4-6). Approx. 21-22% of Kv2.1 and 2.2 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 to 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), Kv2.1KO and Kv2.1/2.2DKO 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), Kv2.1KO and Kv2.1/2.2DKO mice show that Kv5.1 protein levels are severely reduced in Kv2 KO brain. Graph shows quantitation of IP reaction products. Compared to WT mouse brain, the amount of Kv5.1 IPed is decreased by 70% in Kv2.1KO and 95% in Kv2.1/2.2DKO brain samples (one way ANOVA and Tukey’s multiple comparisons test, n=4-5, **** p<0.0001). Thus, Kv5.1 expression is dependent on Kv2 subunits and primarily on Kv2.1.

Figure 4.. Kv5.1 protein is highly expressed in cortex.

(A) Multiplex immunofluorescent labeling of a sagittal section of mouse somatosensory cortex. Kv5.1 (green) immunolabelling varies across cortical layers and is most prominent in layer 2/3, where it is detected in a significant subpopulation of neurons that express Kv2.1 (blue) and/or Kv2.2 (red). Kv5.1 immunolabeling is detected in smaller subpopulations of Kv2 positive neurons in deeper cortical layers. Scale bar = 50 μm. (B) 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 5.. Kv5.1 co-localizes with Kv2 subunits on neuronal somata and proximal dendrites.

(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 (red) 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). Scale bar = 10 μm. (B) In L2/3 neurons, similar PCC values demonstrate that Kv5.1 colocalizes equally with Kv2.1 and Kv2.2 (one way ANOVA and Tukey’s multiple comparisons test, **** p<0.0001, ** p=0.0026, n=33 neurons, 3 WT brains). (C) In L2/3 neurons, ratios of Kv2.1, 2.2 and 5.1 immunolabeling intensity vary between neurons (depicted by each line; n=15 neurons). (D) In L6 cortical neurons, Kv5.1 primarily colocalizes with Kv2.1, as Kv2.2 expression is minimal in this layer. In contrast, in CA3 hippocampal neurons, Kv5.1 primarily colocalizes with Kv2.2, as Kv2.1 expression is low in these neurons. Scale bar = 5 μm.

Fig 6.. Kv5.1 surface expression is significantly reduced in Kv2.1 KO brain.

(A) Immunolabeling for Kv5.1 (green), Kv2.1 (blue) and Kv2.2 (red) in WT (left) and Kv2.1 KO (right) mouse brain sections. Immunolabelling shows that the intensity of Kv5.1 immunolabeling associated with the somatic PM is undetectable or severely reduced in L2/3 neurons in Kv2.1KO brain as compared to wild type (WT). Reduced levels of Kv5.1 remain co-clustered with Kv2.2 in some Kv2.1 KO neurons (as shown in the intensity profile plot). Scale bar = 5 μm. (B) PCC values for Kv2.2 vs. Kv5.1 immunolabelling In L2/3 neurons are significantly reduced in Kv2.1KO brain as compared to WT (one way ANOVA and Sidak’s multiple comparisons test, **** p<0.0001, n=2 pairs of WT and Kv2.1KO brains). (C) Immunolabeling of hippocampal CA3 neurons shows that Kv5.1 is primarily co-clustered with Kv2.2, and that their clustering is preserved in Kv2.1 KO brain. (D) PCCs for Kv2.2 vs. Kv5.1 immunolabelling in CA3 neurons are similar in WT and Kv2.1KO brain (one way ANOVA and Sidak’s multiple comparisons test, **** p<0.0001, n=2).

Fig 7.. Kv5.1 reduces Kv2 channel clustering at ER-PM junctions.

(A) Immunolabelling of L2/3 neurons shows that Kv5.1 (green) is co-clustered with Kv2.1 (blue) and Kv2.2 (red) on a neuronal soma, presumably at sites corresponding to ER-PM junctions. This is evident in the non-uniformity and coincidence of the three labels in the intensity profile (lower right). Scale bar = 2.5 μm. (B) Relative clustering of Kv2 and Kv5.1 subunits was compared by measuring the coefficient of variation of their labeling intensity (CV: SD/mean of pixel intensity). CV values for Kv5.1 were significantly lower than for either Kv2.1 or Kv2.2, suggesting that Kv5.1-containing channels are less clustered (one way ANOVA and Tukey’s multiple comparisons test, **** p<0.0001, ** p=0.0023, n=39 neurons, 2 WT brains). (C) Kv2.1 phosphorylation was assessed by proteomic analysis of Kv2.1 protein immunopurified from brain samples in IPs with Kv2.1 or Kv5.1 antibodies. Left panel. The fraction of spectra containing phosphorylated S/T residues on Kv2.1 was lower in Kv5.1-containing channels compared to 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 to all Kv2.1 channels (one sample t-test, * p=0.03, n=3). (D) Kv2.1BBS was expressed in HEK cells alone or together with Kv5.1 or Kv2.1S586A. Cell surface-expressed Kv2.1BBS was then labelled using AF647-Btx and imaged using TIRF. Kv2.1 is highly clustered when expressed alone but is more diffusely distributed when co-expressed with Kv5.1 or with Kv2.1 S586A. Scale bar = 10 μm. (E) Quantitation of CV values from transfected HEK cells. Co-expression of Kv5.1 and Kv2.1 S586A both resulted in a significant decrease in the CV value of surface Kv2.1BBS labeling, as compared to Kv2.1BBS expressed alone. Thus, Kv2.1/Kv5.1 and Kv2.1/Kv2.1S586A heteromeric channels both have a reduced propensity to cluster at ER-PM junctions (one way ANOVA and Tukey’s multiple comparisons test, **** p<0.0001); n = 21 [Kv2.1], 10 [S586A], 12 [Kv2.1 + Kv5.1], 11 [Kv2.1 + S586A], and 8 [Kv2.1 + GFP] cells).