KCNE4 Antibody

What is KCNE4 and what cellular functions does it regulate?

KCNE4 is a membrane protein belonging to the KCNE family of single transmembrane domain proteins that regulate voltage-gated potassium (Kv) channels. It functions primarily as an inhibitory subunit to certain potassium channels, particularly KCNQ1 and Kv1.3 . KCNE4 plays several key physiological roles:

• In vascular smooth muscle, it regulates Kv7.4 channel function and expression, controlling vascular tone and contractility

• In the immune system, it modulates Kv1.3-dependent processes like T-cell activation, proliferation, and apoptosis

• It appears to be upregulated in lymph nodes during cancer metastasis, potentially affecting chemokine production

KCNE4 modulates potassium channels by altering their biophysical properties, affecting their membrane localization, and changing their responsiveness to physiological stimuli .

What is the molecular weight of KCNE4 and what band sizes should I expect in Western blot?

While KCNE4 has a calculated molecular weight of approximately 18 kDa, it is typically observed at 25-30 kDa in Western blot analyses . This discrepancy is likely due to post-translational modifications. When studying KCNE4 dimerization, you may also detect bands corresponding to dimers (~50-60 kDa) under non-denaturing conditions or when using cross-linking agents .

Which tissues express KCNE4 at detectable levels?

KCNE4 is widely and variably expressed in several human tissues. Based on published research, the highest expression levels are found in:

• Brain, liver, and testis

• Various arteries, particularly mesenteric arteries

• Immune cells, with notable expression in dendritic cells and macrophages

• Heart and skeletal muscle

• Uterus and gastrointestinal tract

Interestingly, KCNE4 expression shows sex-dependent differences, with 2-fold lower expression in female versus male mouse mesenteric arteries .

What are the recommended protocols for KCNE4 antibody in Western blot analysis?

For optimal Western blot detection of KCNE4, follow these guidelines:

Sample preparation:

• For standard detection: Use lysis buffer containing 50 mM boric acid, 100 mM K acetate, 2 mM MgCl₂, 1 mM EGTA, and 1% Triton X-100 (pH 8.5)

• For oligomer detection: Consider using non-denaturing conditions with sample buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol, and 0.2% bromophenol blue

Gel electrophoresis:

• For monomeric KCNE4: 7% SDS-PAGE under denaturing conditions

• For oligomeric forms: 5% acrylamide/bis-acrylamide (30%-0.8% w/v), 0.29 M Tris-HCl pH 8.8, 0.1% SDS

Antibody dilutions:

• Primary antibody: 1:500-1:1000 (e.g., Proteintech 18289-1-AP)

• Secondary antibody: 1:10,000 for fluorescently conjugated secondary antibodies

Detection systems:

• Fluorescence-based systems like Odyssey Infrared Imaging System provide high sensitivity

• Analysis can be performed with software like Image Studio (version 3.0)

How can I optimize immunofluorescence staining for KCNE4?

For optimal immunofluorescence detection of KCNE4:

Fixation and permeabilization:

• Wash cells quickly in appropriate buffer

• Fix with 4% paraformaldehyde for 10 minutes at room temperature

• Wash three times (5 minutes each) with PBS-K⁺

• For intracellular epitopes: Permeabilize with 0.1% Triton X-100 for 20 minutes

Blocking and antibody incubation:

• Block with solution containing 10% goat serum, 5% nonfat milk, and 0.05% Triton X-100 for 60 minutes

• Incubate with primary antibody in 10% goat serum and 0.05% Triton X-100 overnight at 4°C

• Incubate with appropriate fluorophore-conjugated secondary antibodies for 2 hours at room temperature

Imaging recommendations:

• Use confocal microscopy with 63× oil-immersion objective lens (NA 1.32)

• For colocalization studies, quantify using pixel-by-pixel analysis with tools like ImageJ's JACoP (Just Another Colocalization Plugin)

• Calculate Mander's overlap coefficients for quantitative assessment of colocalization

How can I validate KCNE4 antibody specificity in my experimental system?

Multiple validation approaches ensure KCNE4 antibody specificity:

Genetic manipulation approaches:

• Use KCNE4 knockdown with morpholinos or lentiviral shRNA constructs, which should reduce antibody signal

• Compare wild-type tissues with those from KCNE4 knockout models

Heterologous expression systems:

• Compare antibody labeling in cells transfected with KCNE4 versus control vectors

• Use epitope-tagged KCNE4 constructs (HA-tag, GFP fusion) and confirm colocalization with anti-KCNE4 antibody

Peptide competition assays:

• Pre-incubate antibody with immunizing peptide before application to samples

• Signal should be diminished if antibody is specific

Western blot validation:

• Confirm that observed band size matches expected molecular weight (25-30 kDa)

• Verify reduction in band intensity following KCNE4 knockdown

How can I study KCNE4 dimerization and stoichiometry in channel complexes?

KCNE4 can form dimers and associate with potassium channels in different stoichiometries. Several techniques can investigate these complex formations:

Non-denaturing gel electrophoresis:

• Run protein samples on non-denaturing PAGE to preserve protein-protein interactions

• Western blot with anti-KCNE4 antibody can reveal monomeric and dimeric forms

Chemical cross-linking:

• Treat samples with 5 mM dimethyl pimelimidate (DMP) for 1 hour

• Stop reaction with 0.5 M buffer (pH 6.8)

• Compare cross-linked versus non-cross-linked samples by SDS-PAGE

FRET analysis:

• Co-express KCNE4 tagged with donor and acceptor fluorophores (e.g., CFP/YFP)

• Measure energy transfer as indicator of dimerization

• Mutations in the tetraleucine motif (KCNE4(L69-72A)) disrupt dimerization and reduce FRET signals

Single-molecule photobleaching:

• Express KCNE4-loopBAD-GFP in cells

• Use TIRF microscopy to monitor GFP fluorescent spots

• Count bleaching steps to determine subunit composition

• This technique has shown KCNE4 exists in both monomeric and dimeric forms

Chimeric constructs:

• Create KCNE4-Kv1.3 chimeras to force specific stoichiometry

• Test if additional free KCNE4 subunits affect function

• Research indicates a maximum of 4 KCNE4 subunits can associate with a channel complex

How can I investigate calcium-dependent regulation of KCNE4 function?

KCNE4's inhibitory effects on potassium channels are calcium-sensitive, with different inhibition levels at varying calcium concentrations. To study this:

Patch-clamp electrophysiology:

• Control intracellular calcium through patch pipette solutions

• At 10 nM intracellular free Ca²⁺, KCNE4 inhibits KCNQ1 by ~100%

• At 3 nM intracellular free Ca²⁺, inhibition is reduced to ~50%

Calmodulin interaction studies:

• KCNE4 dimerization is calmodulin (CaM)-dependent

• The juxtamembrane tetraleucine motif (L69-72) facilitates CaM-dependent interactions

• Use co-immunoprecipitation with anti-calmodulin antibodies at different Ca²⁺ concentrations

Mutagenesis:

• Generate KCNE4 mutants disrupting the tetraleucine motif (KCNE4(L69-72A))

• These mutants show impaired dimerization in co-immunoprecipitation, non-denaturing PAGE, and FRET assays

Calcium imaging combined with immunolocalization:

• Monitor KCNE4 localization changes following calcium flux

• Correlate with functional changes in channel activity

What approaches can reveal KCNE4's tissue-specific functions?

KCNE4 exhibits tissue-specific and sex-dependent expression and function. To investigate these aspects:

Comparative expression analysis:

• Use quantitative PCR and Western blot to measure KCNE4 levels across tissues

• In mesenteric arteries, KCNE4 expression is 2-fold lower in females versus males

Proximity ligation assay (PLA):

• Detect co-localization of KCNE4 with tissue-specific binding partners

• Successfully used to show KCNE4 co-localization with Kv7.4 in mesenteric artery myocytes

Sex-specific functional differences:

• In Kcne4 knockout mice, mesenteric artery contractility increases in males but not females

• Responses to Kv7.2-7.5 channel activator ML213 decrease in males but not females

• Vasorelaxation responses to isoprenaline decrease in both sexes

Comparative localization studies:

• Use immunohistochemistry to determine subcellular localization in different tissues

• In vascular smooth muscle, KCNE4 knockdown reduces membrane expression of Kv7.4

How is KCNE4 involved in vascular physiology and potential pathologies?

KCNE4 plays crucial roles in vascular function:

Regulation of vascular tone:

• KCNE4 co-localizes with Kv7.4 in mesenteric arteries

• Knockdown of KCNE4 leads to:

  • Reduced Kv7.4 membrane abundance

  • Depolarized membrane potential

  • Augmented response to vasoconstrictors

Quantitative effects on vasoconstriction:

• KCNE4 knockdown increases sensitivity to methoxamine

• EC₅₀ decreases from 5.7 ± 0.63 μM to 1.6 ± 0.23 μM

Sex-dependent vascular effects:

• Kcne4 deletion increases contractility in response to α-adrenoceptor agonist methoxamine in male but not female mice

• Decreased responses to Kv7.2-7.5 channel activator ML213 in males only

• Decreased vasorelaxation to isoprenaline in both sexes

Molecular mechanisms:

• Kv7.4 protein expression in females is twice that in males

• Kcne4 deletion reduces Kv7.4 expression in both sexes

These findings suggest targeting KCNE4 could be relevant for vascular disorders with sex-specific prevalence patterns.

What role does KCNE4 play in immune cell function and potential immunological disorders?

KCNE4 has significant functions in the immune system:

Expression patterns:

• Unlike T cells, antigen-presenting cells (APCs) like dendritic cells and macrophages express notable levels of KCNE4

Functional effects in immune cells:

• KCNE4 modulates Kv1.3-related events in leukocyte physiology

• It regulates several immunological processes:

  • Delocalization from the immunological synapse

  • IL-2 production in T-cells

  • APC activation

  • Cell proliferation and apoptosis

Experimental observations:

• KCNE4 knockdown in CY15 dendritic cells results in:

  • Increased cell size

  • Slightly higher proliferation rates

• KCNE4 completely inhibits Kv1.3 current in Xenopus oocytes and HEK293 cells

• It causes 2-fold inhibition, 3-fold slowed activation, and 2-fold speeded inactivation of Kv1.3 in HEK293 cells

These findings position KCNE4 as a potential therapeutic target for Kv1.3-related immunological disorders.

What is the emerging evidence for KCNE4's role in cancer progression?

Recent research has identified potential roles for KCNE4 in cancer:

Lymph node metastasis:

• KCNE4 is upregulated in submandibular lymph nodes (SLNs) with metastatic melanoma

• Confirmed by microarray analysis, real-time PCR, and immunohistochemistry

Functional implications in cancer progression:

• KCNE4 expression in lymphatic endothelial cells upregulates:

  • Ccl17 and Ccl19 chemokines involved in melanoma metastasis

  • Mmp3 matrix metalloproteinase

Clinical relevance:

• KCNE4 expression has been detected in human lymph nodes with metastatic melanoma

• This suggests KCNE4 could be a biomarker or therapeutic target in metastatic disease

Further research is needed to fully elucidate KCNE4's role in cancer progression and its potential as a therapeutic target in oncology.

How do I resolve multiple bands or unexpected molecular weights in KCNE4 Western blots?

Multiple bands in KCNE4 Western blots may represent different forms or non-specific binding:

Potential causes and solutions:

Validation approaches:

• Compare with positive and negative control tissues/cells

• Use KCNE4 knockdown or overexpression to identify specific bands

• If studying dimerization, use cross-linking with DMP (5 mM for 1 hour) to stabilize dimers

• Run non-denaturing gels in parallel to identify oligomeric states

What approaches can enhance detection of low abundance KCNE4 expression?

For tissues with low KCNE4 expression, several techniques can improve detection:

Sample enrichment:

• Immunoprecipitate KCNE4 before Western blotting

• Use membrane fraction enrichment protocols

• Scale up starting material (increase protein loading)

Signal amplification:

• Use high-sensitivity detection systems (e.g., Odyssey Infrared Imaging System)

• Try tyramide signal amplification for immunohistochemistry

• Consider proximity ligation assay (PLA) for detecting KCNE4 interactions

Alternative detection methods:

• Use quantitative PCR to confirm expression at mRNA level first

• Consider RNAscope in situ hybridization for tissue localization of mRNA

• For functional studies in tissues with low expression, use pharmacological tools that target KCNE4-modulated channels

Technical optimization:

• Extend primary antibody incubation time (overnight at 4°C)

• Optimize antibody concentration through titration

• Use high-sensitivity substrates for chemiluminescence detection

How do I distinguish between specific and non-specific binding in immunohistochemistry?

Ensuring specificity in KCNE4 immunohistochemistry:

Essential controls:

• Omit primary antibody (secondary antibody control)

• Use tissues from KCNE4 knockout models as negative controls

• Include tissues with known high KCNE4 expression as positive controls (brain, liver, testis)

• Pre-absorb antibody with immunizing peptide to confirm specificity

Validation strategies:

• Compare staining pattern with in situ hybridization results

• Verify with multiple antibodies targeting different KCNE4 epitopes

• Correlate with functional data (e.g., effects of KCNE4 knockdown)

Pattern interpretation:

• KCNE4 should show both membrane and intracellular (ER, Golgi) staining

• In vascular tissue, look for co-localization with Kv7.4 in smooth muscle cells

• In lymph nodes, examine endothelial cells for KCNE4 expression

Technical considerations:

• Optimize antigen retrieval methods

• Test multiple fixation protocols

• Consider tyramide signal amplification for low abundance expression