Journal: Journal of Biological Chemistry

Article Title: A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

doi: 10.1074/jbc.ra118.006549

Figure Lengend Snippet: Figure 5. 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 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) while WT KChIP2a1 did not (p = 0.6970) as also shown in (b). Error bars represent mean +/- SEM. * = 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. 24

Article Snippet: Kv4 auxiliary subunit expression was carried out using Human DPP6 (RC216919), Human KChIP1a (RC224442), 1b (RC208255), 2a1 (RC213131), 2b (RC203823), 3a (RC203957), 4bL (RC211488), 4a (RC211613), and Mouse KChIP2a (MC211934) were obtained by guest on January 8, 2019 http://w w w .jbc.org/ D ow nloaded from Ca2+ regulation of KChIP-Kv4 channel complexes from Origene.

Techniques: Sequencing, Mutagenesis, Construct, Expressing, Comparison

Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 6. DAPA alleviated ion channel disorders and atrial fibrosis caused by Ang II. A. Immunoblotting analysis of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40, smad2/3, p-smad2/3, TGF-β1 in atrial tissue. B, C. Quantification of CAMKII, ox-CAMKII expression level in atrial tissue. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. D-I. Quantification of atrial electrical activity associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J, K. Quantification of atrial fibrosis associated protein expression level. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal-Wallis post hoc multicomparison test (B–K). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Western Blot, Expressing, Activity Assay

Journal: European journal of pharmacology

Article Title: Dapagliflozin: A sodium-glucose cotransporter 2 inhibitor, attenuates angiotensin II-induced atrial fibrillation by regulating atrial electrical and structural remodeling.

doi: 10.1016/j.ejphar.2024.176712

Figure Lengend Snippet: Fig. 7. SGLT2i alleviated ion channel disorders and atrial fibrosis caused by activation of CAMKII. A-I Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, Nav1.5, Kv4.3, Kv4.2, Kchip2, Kir2.1, Cx40 in HL-1 atrial myocytes. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. J-N. Immunoblotting analysis and quantitation of the protein expression of CAMKII, ox-CAMKII, smad2/3, p-smad2/3, TGF-β1 in primary mouse fibroblast. n = 4. *p < 0.05 vs. CTL group and #p < 0.05 vs. Ang II group. Statistical significance was assessed by one-way ANOVA analysis followed by Tukey tests or Kruskal- Wallis post hoc multicomparison test (B–I and K–N). Data are presented as means ± SEM.

Article Snippet: Protein lysates of tissue or cells were separated by 8% SDS-PAGE and transferred to NC membrane, and blocked for 2 h using 5% non-fat milk and probed with primary polyclonal antibodies against CaMKII (Abclonal), oxCaMKII (GeneTex), Nav1.5 (Abmart), Kv4.3 (Abclonal), Kv4.2 (Cell Signaling Technology), Kchip2 (Abclonal), Kir2.1(Zenbio), Cx40 (GeneTex), TGF-β1(Santa cruz), smad (Zenbio), p-smad (Zenbio) at 4 ◦C overnight.

Techniques: Activation Assay, Western Blot, Quantitation Assay, Expressing

Journal: Oncotarget

Article Title: A novel KCND3 mutation associated with early-onset lone atrial fibrillation

doi: 10.18632/oncotarget.23303

Figure Lengend Snippet: (A) Typical currents recorded from HEK293 cells transfected WT or T361S of K v 4.3 together with KChIP2 in response to increasing step potential. (B) Current-voltage relationship. The protocol was shown in the inset. Data was shown as means ± SEM.

Article Snippet: T vectors containing cDNA encoding human KChIP2 (NM_173192.2) or human KCND3 (NM_172198.2) were purchased from Proteintech Company (Wuhan, China).

Techniques: Transfection

Journal: Oncotarget

Article Title: A novel KCND3 mutation associated with early-onset lone atrial fibrillation

doi: 10.18632/oncotarget.23303

Figure Lengend Snippet: (A) Cell surface biotinylation assays were performed in Hek293 cells co-transfected KChIP2 with wild type or mutant T361S of K v 4.3 after 48 hours. Intergrin α5 was used as loading to calibrate the cell-surface proteins. No GAPDH was detected in the biotinylated fraction (data not shown). (B) The total expression of K v 4.3 protein was extracted from HEK293 cells co-transfected KChIP2 with wild type or mutant T361S of K v 4.3. This experiment was repeated at least three times, and similar results were obtained. Data was shown as means ± SEM. * P < 0.05.

Article Snippet: T vectors containing cDNA encoding human KChIP2 (NM_173192.2) or human KCND3 (NM_172198.2) were purchased from Proteintech Company (Wuhan, China).

Techniques: Transfection, Mutagenesis, Expressing

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, Comparison

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 4 Effects of IL-17 knockout on the Ito and KChIP2 expression in the hearts of diabetic mice. a Representative traces of the Ito. b Current density-voltage (I–V) relationship of the Ito. n = 8–11 cells. c Current density-voltage (I–V) relationship of the Iss. n = 9–16 cells. d Mean membrane capacitance of the Ito and Iss n = 9–16 cells. e The protein and mRNA levels of Kv4.2. n = 7. f The protein and mRNA levels of Kv4.3. n = 7. g The protein and mRNA levels of KChIP2. n = 5. *P < 0.05 vs. WT mice; #P < 0.05 vs. WT+DM mice. WT wild-type, IL-17 KO IL-17 knockout, WT+DM wild-type+diabetes mellitus, IL-17 KO+DM IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing, Membrane

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 8 Knockout of IL-17 protects against ventricular arrhythmias in STZ-induced diabetic mice. Knockout of IL-17 downregulates the expression of NF-κB, which suppresses the expression of KCNIP2, which encodes potassium voltage-gated channel interacting protein 2; CACNA1C, which encodes the pore-forming subunit of the voltage-gated L-type calcium channel Cav1.2; and SCN5A, which encodes the pore-forming subunit of the voltage-gated sodium channel Nav1.5. Decreased expression of KChIP2 and Nav1.5 prolonged the APD and slowed the conduction velocity, which increased susceptibility to ventricular arrhythmias. KCNIP2, potassium voltage-gated channel interacting protein 2; CACNA1C, calcium voltage-gated channel subunit alpha 1 C; KCND2, potassium voltage-gated channel subfamily D member 2; KCND3, potassium voltage-gated channel subfamily D member 3; SCN5A, sodium voltage-gated channel alpha subunit 5; WT+DM, wild-type+diabetes mellitus; IL-17 KO+DM, IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing