KCNH7 Antibody

What is KCNH7 and why is it significant for neuroscience research?

KCNH7 (also known as ERG3 or Kv11.3) is a member of the voltage-gated potassium (K+) channel subfamily H. It is a pore-forming (alpha) subunit of voltage-gated potassium channels primarily expressed in the brain, particularly in neurons of the cortex, hippocampus, and cerebellum .

The significance of KCNH7 lies in its role in regulating neuronal excitability. Research has shown that KCNH7 channels stabilize the resting membrane potential and dampen spontaneous activity in cerebellar Purkinje cells and hippocampal CA1 neurons . This function makes KCNH7 a potential therapeutic target for various neurological and psychiatric disorders.

Studies have associated KCNH7 variants with:

• Schizophrenia treatment responses (particularly risperidone efficacy)

• Bipolar spectrum disorder

• Pediatric epilepsy

• Depressive-like behaviors

What experimental applications are validated for KCNH7 antibodies?

Based on the available research data, KCNH7 antibodies have been validated for multiple experimental applications:

When selecting an antibody for your application, consider the specific validation data. For instance, Proteintech's 13622-1-AP has been validated for WB (1:500-1:1000 dilution), IP (0.5-4.0 μg for 1.0-3.0 mg total protein), and IHC (1:20-1:200 dilution) .

How should I store and handle KCNH7 antibodies to maintain optimal activity?

Proper storage and handling of KCNH7 antibodies is crucial for maintaining their activity and specificity:

Storage conditions:

• Store at -20°C for most commercially available KCNH7 antibodies

• Some recombinant antibodies require -80°C storage

• Avoid repeated freeze-thaw cycles

• Most preparations are stable for 12 months when stored properly

Buffer compositions:

• Most KCNH7 antibodies are supplied in PBS with stabilizers:

  • PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

  • Phosphate buffered solution, pH 7.4, with 0.05% stabilizer and 50% glycerol

  • Some antibodies are provided in PBS only (BSA and azide free) for conjugation purposes

Aliquoting recommendation:
For the 13622-1-AP antibody, aliquoting is noted as unnecessary for -20°C storage, but for other antibody preparations, dividing into small aliquots is recommended to prevent degradation from repeated freeze-thaw cycles .

How can I validate KCNH7 antibody specificity in knockout models?

Validating antibody specificity using knockout models is critical for ensuring reliable experimental results. Based on the research literature, here is a methodological approach:

Step 1: Generate appropriate knockout models
Researchers have successfully used CRISPR-Cas9 to create global KCNH7 knockout mice by targeting exon 5 of the KCNH7 gene . For conditional knockouts, a floxed approach (as used for KCNH2) can be adapted .

Step 2: Genotyping confirmation
PCR verification using specific primers:

• Forward primer: GTA GAG ACT CCG TGG ATC ATT TCA TAT AGG TA

• Reverse primer: CCA AGT ATG ATG AAT AGC TCA GTA ATT ATT TCA GAG CA

• WT-specific primer: GTT TGA ATC TGA TGT GGA TCC CAG C

Step 3: Protein-level validation
Immunoprecipitation with an ERG3-specific antibody using brain lysate has been shown to effectively demonstrate absence of the protein in knockout tissue . Western blot analysis of hippocampus and cerebellum extracts can further confirm knockout at the protein level.

Step 4: Functional validation
Electrophysiological recordings comparing wild-type and knockout neurons can provide functional validation of the knockout model and antibody specificity .

A critical control demonstrated in the literature is the comparison of immunostaining patterns between:

• Wild-type (+/+) tissue (positive signal)

• Heterozygous (+/-) tissue (reduced signal)

• Homozygous knockout (-/-) tissue (absent signal)

What are the optimal immunohistochemistry protocols for detecting KCNH7 in brain tissue sections?

Detecting KCNH7 in brain tissue requires careful attention to several methodological variables:

Tissue preparation:

• Fresh frozen sections are preferable for preserving antigenicity

• If using paraffin-embedded sections, antigen retrieval is crucial

Antigen retrieval methods:

• TE buffer pH 9.0 is suggested as the primary retrieval method

• Alternatively, citrate buffer pH 6.0 can be used

Blocking and antibody incubation:

• Block with 5-10% normal serum (matching the secondary antibody host) and 0.1-0.3% Triton X-100

• Primary antibody dilutions:

  • For IHC: 1:20-1:200 (optimization recommended for each tissue type)

• Incubate overnight at 4°C for optimal signal-to-noise ratio

Detection systems:

• For fluorescent detection: compatible with standard fluorophore-conjugated secondary antibodies

• For chromogenic detection: HRP/DAB systems have been successfully used

Control considerations:

• Negative controls: secondary antibody only; isotype control

• Positive controls: human brain tissue, mouse brain tissue (particularly cerebellum and hippocampus)

Note on regional expression: KCNH7 expression is particularly high in cerebellar Purkinje cells and hippocampal neurons, making these regions ideal for positive controls and expression studies .

How can experimental conditions be optimized to detect KCNH7 variants associated with neuropsychiatric disorders?

Several KCNH7 variants have been associated with neuropsychiatric disorders, requiring specific experimental approaches for their detection and characterization:

Key variants of interest:

• c.1181G>A (p.Arg394His) - associated with bipolar spectrum disorder

• c.83A>G (p.K28R), c.1919A>G (p.E640G), c.1324C>T (p.R442X) - associated with pediatric epilepsy

• rs77699177 (C>T, intronic) - associated with risperidone response in schizophrenia

Methodological approaches:

What controls are essential when using KCNH7 antibodies to distinguish between other ERG family members?

Distinguishing KCNH7 (ERG3) from other ERG family members (particularly ERG1/KCNH2 and ERG2) requires rigorous controls:

Sequence homology considerations:
The ERG family shares significant homology in several domains, particularly:

• The pore-forming region

• The cyclic nucleotide-binding homology domain (CNBHD)

• The PAS (Per-Arnt-Sim) domain

Essential controls:

• Recombinant protein controls:

  • Purified recombinant KCNH7, KCNH2, and other related potassium channels

  • Test antibody cross-reactivity against all family members

• Genetic models:

  • KCNH7-specific knockout tissues (as negative controls)

  • Conditional KCNH2 knockout tissues (to verify specificity)

  • siRNA or shRNA knockdown models for KCNH7

• Antibody epitope information:

  • Verify that the antibody targets a unique region of KCNH7

  • Commercial antibodies like Proteintech's 13622-1-AP are generated against KCNH7 fusion protein Ag4532

• Western blot verification:

  • KCNH7 has an observed molecular weight of 135 kDa

  • Compare migration patterns with other ERG family members

• Expression pattern controls:

  • ERG1 (KCNH2) is highly expressed in cardiomyocytes

  • ERG3 (KCNH7) is primarily expressed in neurons

  • Tissue-specific expression patterns can help verify specificity

How can I optimize Western blot protocols for detecting native KCNH7 in brain tissue samples?

Detecting native KCNH7 in brain samples presents several technical challenges. Here is an optimized protocol based on published research:

Tissue extraction and sample preparation:

• Homogenize fresh brain tissue in ice-cold buffer containing:

  • 150 mM NaCl

  • 50 mM Tris-HCl, pH 7.5

  • 5 mM EDTA

  • 1% NP-40 or 1% Triton X-100

  • Complete protease inhibitor cocktail

• Use 12 strokes with a Potter homogenizer

• Centrifuge at 1000g for 10 min at 4°C

• Mix supernatant with loading dye containing:

  • 500 mM DTT

  • 0.05% bromophenol blue

  • 50% glycerol

  • 10% SDS

  • 250 mM Tris-Cl (pH 6.8)

• Denature at 97°C for 5 minutes

Gel electrophoresis and transfer conditions:

• Use 6-8% polyacrylamide gels due to KCNH7's large size (135 kDa)

• Transfer to PVDF membrane at low current (30V) overnight at 4°C

Antibody incubation:

• Block with 5% non-fat dry milk in TBST

• Primary antibody dilution: 1:500-1:1000

• Incubate overnight at 4°C

Region-specific considerations:

• Hippocampus and cerebellum show highest KCNH7 expression

• Both young and adult mice tissue samples have been successfully used

Validated antibodies for Western blot:
Several antibodies have been validated in the literature:

• Proteintech #13622 (RRID: AB_10638620)

• Alomone #APC112 (RRID: AB_2039937)

• Thermo Fisher Scientific #PA5-68276 (RRID: AB_2691815)

What methodologies are most effective for studying KCNH7's role in neuronal excitability?

Research on KCNH7's role in neuronal excitability requires a combination of molecular, cellular, and electrophysiological approaches:

Electrophysiological methods:

• Acute slice preparation:

  • Prepare 250 μm cerebellar or hippocampal slices in ice-cold carbonated (95% O₂, 5% CO₂) sucrose slicing solution

  • Incubate in standard artificial cerebrospinal fluid (ACSF) at 35°C for 1 hour, then at room temperature

  • Use within 8 hours after incubation

• Patch-clamp recordings:

  • Whole-cell voltage-clamp recordings to measure KCNH7 currents

  • Current-clamp recordings to assess effects on neuronal excitability

  • Depolarization protocols to elicit KCNH7 currents (characteristic diminishing currents with depolarization >20 mV)

Pharmacological approaches:

• KCNH7 channel blockers:

  • E-4031 (10 μM) - shown to depolarize Purkinje cells by 2-5 mV

  • Terfenadine (30 μM)

Genetic models:

• Global knockout approach:

  • CRISPR-Cas9 deletion of exon 5 of KCNH7

  • Assessment of hyperexcitability in cerebellar Purkinje cells

• Conditional knockout approach:

  • Cell-type specific deletion using Cre-loxP system

  • L7(Pcp2)-Cre for Purkinje cell-specific deletion

Behavioral assessments:
For correlating channel function with behavior:

• Forced swim test and tail suspension test (for depressive-like behaviors)

• Seizure susceptibility tests (PTZ-induced seizures)

• Startle response measurements

Molecular mechanisms:
Studies have shown that KCNH7 knockdown leads to:

• Enhancement of neuronal intrinsic excitability

• Increased seizure susceptibility

• Depressive-like behaviors

How can KCNH7 antibodies be used to investigate pharmacogenomic associations in schizophrenia?

Research has identified significant associations between KCNH7 variants and antipsychotic treatment responses, particularly with risperidone. Here's a methodological approach to investigate these associations:

Study design considerations:

• Cohort selection: 393 schizophrenia patients treated with risperidone for 6 weeks has proven effective

• Outcome measures: Positive and Negative Syndrome Scale (PANSS) score reduction rates

• Genotype analysis: Focus on specific SNPs including rs77699177 (C>T) and rs2241240

Experimental approach:

• Patient stratification by genotype:
  For rs77699177, patients can be stratified into:

  • CC genotype group

  • TC genotype group

  Research shows significant differences in treatment response:

  Genotype	PANSS Reduction Rate (Mean ± SD)	P-value
  CC	55.8 ± 23.0	Baseline
  TC	70.9 ± 20.3	0.000110

• Protein expression analysis:

  • Compare KCNH7 protein expression levels between genotype groups using antibody-based methods

  • Western blot of peripheral blood lymphocytes or post-mortem brain tissue

  • Correlate protein expression with treatment response

• Functional characterization:

  • Electrophysiological recordings to assess channel function in cell models expressing different variants

  • Assessment of risperidone effects on channel currents

• Translational applications:

  • Development of predictive biomarkers for risperidone response

  • Potential for KCNH7 as a therapeutic target to improve treatment response

What are the technical considerations for studying KCNH7 in relation to bipolar spectrum disorder?

The research associating KCNH7 variants with bipolar spectrum disorder presents several technical considerations for further investigation:

Key variant focus:
The c.1181G>A (p.Arg394His) variant has shown the strongest association with bipolar spectrum disorder . This variant:

• Was carried by all 14 subjects from families with prevalent bipolar disorder

• Had the highest enrichment among individuals with bipolar spectrum disorder (χ² = 7.3)

• Showed the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031), and major affective disorder (P = 0.016)

Methodological approaches:

How can I detect and characterize KCNH7 in pediatric epilepsy research?

Recent research has identified KCNH7 as a candidate gene in pediatric epilepsy. Here's a methodological approach to study this association:

Genetic screening approach:

• Trio whole exome sequencing in pediatric epilepsy cohorts has successfully identified de novo KCNH7 variants

• For large cohorts, consider screening potential candidate genes after initial negative results

Key variants to investigate:
Three de novo variants have been associated with pediatric epilepsy:

• c.83A>G/p.K28R

• c.1919A>G/p.E640G

• c.1324C>T/p.R442X (nonsense mutation)

Pathogenicity assessment:

• Compare variant frequencies between patient cohorts and controls in population databases (e.g., gnomAD)

• Use pathogenicity prediction tools: Mutation Taster, PolyPhen2, SIFT

• Assess conservation of affected residues across species

Phenotype-genotype correlation approach:
The severity of epilepsy symptoms appears to correlate with the extent of the mutations' impact on protein structure:

• p.K28R: Associated with seizure onset at 15 months, good response to levetiracetam, no developmental delay

• p.E640G: Associated with West syndrome, seizure onset at 5 months, initial developmental regression

• p.R442X (nonsense): Associated with seizure onset at 8 months, brain MRI abnormalities, developmental regression

Expression analysis considerations:
Analysis of BrainSpan transcriptomic data indicates that KCNH7 gene expression:

• Peaks around one year of age

• Remains relatively lower in subsequent years
  This pattern may explain the early onset of seizures and the favorable prognosis observed in patients .

What immunofluorescence techniques are optimal for localizing KCNH7 in specific neuronal populations?

Immunofluorescence localization of KCNH7 in neuronal populations requires specific technical considerations:

Tissue preparation:

• Perfusion fixation with 4% paraformaldehyde is preferred

• Post-fixation should be kept to a minimum (2-4 hours) to preserve antigenicity

• Cryoprotection in sucrose followed by sectioning at 20-40 μm thickness

• Optimal sections: cerebellar slices (for Purkinje cells) and hippocampal slices (for CA1 neurons)

Antigen retrieval:

• TE buffer pH 9.0 is recommended

• Alternatively, citrate buffer pH 6.0 can be used

• Heat-induced epitope retrieval (HIER) at 95-98°C for 15-20 minutes

Blocking and permeabilization:

• 10% normal serum (matching secondary antibody host)

• 0.3% Triton X-100 in PBS

• Block for 1-2 hours at room temperature

Antibody selection and dilution:

• Primary antibody dilutions for immunofluorescence: 1:20-1:200

• Validated antibodies: Proteintech #13622-1-AP, Alomone #APC112

• For colocalization studies, select antibodies raised in different host species

Counterstaining options:

• Neuronal markers: NeuN, MAP2, or βIII-tubulin

• Cerebellar Purkinje cell markers: Calbindin

• Nuclear counterstain: DAPI

• For synaptic localization: Synapsin-1, PSD-95

High-resolution imaging recommendations:

• Confocal microscopy with optical sections of 0.5-1 μm

• Super-resolution techniques (e.g., STED, STORM) for synaptic localization

• Z-stack acquisition for 3D reconstruction of neuronal morphology

Expected localization pattern:
KCNH7 is expressed in:

• Cerebellar Purkinje cells

• Hippocampal CA1 neurons

• Cerebral cortex neurons