kchip2b  (OriGene)

Journal: eLife

Article Title: Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons

doi: 10.7554/elife.40818

Figure Lengend Snippet: Figure 3. Functional upregulation of Kv4 channels depended upon KChIPs. (a) Ca2+ transients in response to bAPs (arrows) at proximal and distal dendritic spines in iSPNs from BACHD mice with the internal perfusion of KChIP2 antibody (1:50); perfusion rescued dendritic excitability in HD iSPNs. (b) The dendritic index was significantly smaller with internal perfusion of the denatured KChIP2 antibody (boiled) (p=0.0022, Mann-Whitney U, Two- Tailed, n = 6). (c) Voltage responses to optogenetic stimulation of corticostriatal terminals at distal dendrites in iSPNs from BACHD mice with internal perfusion of KChIP2 antibody before and after 4-AP (2 mM). (d) The Kv4 index was significantly reduced by antibody perfusion (p=0.0006, Mann- Whitney U, Two-Tailed, n = 7). In all the dialysis experiments, recordings were taken >30 min after entering whole cell mode. (e) Western blot of Kv4.2 from striatum homogenates. (f) Kv4.2 protein levels show no significant difference between WT and Q175 ±mice (p=0.061, Mann-Whitney U, Two- Tailed, n = 5. (g) Co-immunoprecipitation of Cav3.2 channels with Kv4.2 channels from mouse striatum (Kv4.2 pulldown). (h) Voltage responses to optogenetic stimulation of corticostriatal terminals at distal dendrites of iSPNs from BACHD mice in the presence of the Cav3 Ca2+ channel blocker mibefradil (1 mM) before and after 4-AP (2 mM). (i) Changes in the Kv4 index were consistent with the loss of dendritic excitability in iSPNs being mediated by the interaction of Cav3 Ca2+ channels with Kv4.2 channels through KChIPs (p=0.0006, Mann-Whitney U, Two-Tailed, n = 7. See Figure 3— source data 1. DOI: https://doi.org/10.7554/eLife.40818.015 The following source data and figure supplements are available for figure 3:

Article Snippet: The KChIP2 antibody (Neuromab 75–004) also was diluted at the same 1:50 ratio.

Techniques: Functional Assay, MANN-WHITNEY, Two Tailed Test, Western Blot, Immunoprecipitation

Journal: eLife

Article Title: Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons

doi: 10.7554/elife.40818

Figure Lengend Snippet: Figure 4. TrkBR signaling regulated Kv4 channel association with KChIPs. (a) KChIPs co-immunoprecipitated with Kv4.2 in mice striata, KChIP1 and KChIP2 more robustly associated with Kv4.2 than KChIP3/4. To the right is a schematic of the Kv4.2/KChIP/Cav3.2 membrane complex. (b) Detection of Kv4.2 phosphorylation at Thr607 and Thr602, P-Kv4.2 levels were normalized to total Kv4.2; in Q175 ±mice, Kv4.2 phosphorylation was decreased at both Thr607 (p=0.03078, Mann-Whitney U, Two-Tailed, n = 6) and Thr602 (p=0.0226, Mann-Whitney U, Two-Tailed, n = 6). (c) Co-immunoprecipitation of KChIP2 with Kv4.2 in WT striata after incubation with BDNF or with vehicle control; association of KChIP2 was decreased (p=0.01208, Mann-Whitney U, Two-Tailed, n = 5). (d) Co-immunoprecipitation of KChIP2 with Kv4.2 in Q175 ±striata after incubation with BDNF or with vehicle control; no difference in Kv4.2 association with KChIP2 (p=0.83366, Mann-Whitney U, Two-Tailed, n = 5). (e) Co-immunoprecipitation of KChIP2 with Kv4.2 in Q175 ±striata after incubation with ROCKi +BDNF or with vehicle control; association of KChIP2 with Kv4.2 was decreased (p=0.03662, Mann-Whitney U, Two-Tailed, n = 5). (f) Diagram of the proposed mechanism showing how TrkBR and p75NTR signaling modulate KChIP association with Kv4.2 and Kv4.2 channel gating. See Figure 4—source data 1. DOI: https://doi.org/10.7554/eLife.40818.024 The following source data and figure supplements are available for figure 4:

Article Snippet: The KChIP2 antibody (Neuromab 75–004) also was diluted at the same 1:50 ratio.

Techniques: Immunoprecipitation, Membrane, Phospho-proteomics, MANN-WHITNEY, Two Tailed Test, Incubation, Control

Journal: The Journal of Biological Chemistry

Article Title: A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca 2+ regulation of Kv4 channels

doi: 10.1074/jbc.RA118.006549

Figure Lengend Snippet: Ca2+ regulation of Kv4.2–KChIP2 complexes is KChIP isoform-dependent. a, consensus protein domain organization of KChIP family members (top). Protein domain organization of four KChIP2 isoforms that demonstrates N-terminal variability (below). b, two-way ANOVA returned significant differences in peak Kv4.2 current density between KChIP2 isoforms (p = 0.0005), by treatment (p = 0.0227), and the interaction (p = 0.0150). However, Kv4.2 peak current density was unaffected by intracellular Ca2+ when expressed with long forms of KChIP2 (KChIP2a1, p = 0.9415; KChIP2a, p = 0.9997), whereas Kv4.2 peak current was significantly increased in the presence of Ca2+ for shorter forms of KChIP2 (KChIP2b, p = 0.0114; KChIP2c, p = 0.0203). c, sequence alignment of human N-terminal domains of KChIP2 isoforms. The putative polybasic domain conserved in Ca2+-insensitive KChIP isoforms is underlined, and basic residues are indicated with an asterisk. d, site-directed acidification of the putative polybasic motif in KChIP2a1 rescues Ca2+ enhancement of peak current density. Two-way ANOVA returned no differences between WT KChIP2a1 and mutant KChIP2a1 groups by construct expression (p = 0.1419) or treatment (p = 0.6426); however, the interaction was significant (p = 0.0124) likely due to differences in peak current density between WT and mutant KChIP2a1. Sidak's multiple comparison revealed that mutant KChIP2a1 responded to Ca2+ (p = 0.0107), whereas WT KChIP2a1 did not (p = 0.6970) as also shown in b. Error bars, mean ± S.E. *, p < 0.05; ***, p < 0.001 by two-way ANOVA and Sidak's multiple comparison test. Refer to Table 2 for numerical data and replicate information.

Article Snippet: Kv4 auxiliary subunit expression was carried out using human DPP6 (RC216919), human KChIP1a (RC224442), KChIP1b (RC208255), KChIP2a1 (RC213131), KChIP2b (RC203823), KChIP3a (RC203957), KChIP4bL (RC211488), KChIP4a (RC211613), and mouse KChIP2a (MC211934) (Origene).

Techniques: Sequencing, Mutagenesis, Construct, Expressing