KCNJ10 Antibody

What is KCNJ10 and what are its key structural features?

KCNJ10, also known as Kir4.1 or SeSAME, is an inwardly-rectifying potassium channel, subfamily J, member 10, encoded by the KCNJ10 gene in humans. This ATP-dependent potassium channel consists of three transmembrane domains, with the pore-forming motif located within the second transmembrane domain . The protein has a molecular weight of approximately 42-43 kDa and functions as an ATP-dependent potassium channel .

KCNJ10 channels are characterized by their greater tendency to allow potassium to flow into the cell rather than out of it. Their voltage dependence is regulated by extracellular potassium concentration; as external potassium increases, the voltage range for channel opening shifts toward more positive voltages . The inward rectification is primarily due to blockage of outward current by internal magnesium, and the channel can be blocked by extracellular barium and cesium .

What are the primary physiological roles of KCNJ10 in different tissues?

KCNJ10 serves critical functions in multiple tissues:

• Brain: Responsible for potassium buffering action of glial cells, serving as a cellular marker for characterizing astrocytes .

• Inner Ear: Expressed in the stria vascularis, particularly in intermediate cells, where it contributes to maintaining the endocochlear potential critical for normal hearing .

• Kidney: In collaboration with KCNJ16, mediates basolateral K+ recycling in distal convoluted tubules, a process essential for Na+ reabsorption .

Mutations in KCNJ10 cause an autosomal recessive disease known as EAST syndrome or SeSAME syndrome (OMIM 612780), characterized by epilepsy, ataxia, sensorineural deafness, mental retardation, and electrolyte imbalances .

What criteria should guide KCNJ10 antibody selection for research applications?

When selecting KCNJ10 antibodies, consider these critical factors:

• Antibody Format: Available as polyclonal (higher sensitivity, multiple epitope recognition) or monoclonal (higher specificity, consistent results) .

• Species Reactivity: Most KCNJ10 antibodies show reactivity against human, mouse, and rat proteins. Some have predicted reactivity against additional species such as pig, bovine, horse, sheep, rabbit, dog, and Xenopus .

• Application Compatibility: Verify validation for your specific application (WB, IHC, ICC, IF) .

• Immunogen Information: Check the immunogen used (typically synthetic peptides derived from C-terminal or mid-protein regions of human KCNJ10) .

• Validation Data: Review images of expected staining patterns and molecular weight detection (typically ~42-43 kDa) .

How can I validate the specificity of a KCNJ10 antibody in my experimental system?

Comprehensive validation includes multiple complementary approaches:

• Genetic Controls: The gold standard involves using Kcnj10 knockout tissues. As noted in one study, "The absence of signal in Kcnj10 knockout mice verified the specificity of the antibody" .

• Peptide Competition: Pre-incubate the antibody with the immunizing peptide and run parallel with non-blocked antibody; specific signals should be reduced or eliminated.

• Expression Systems: Compare staining in cells with endogenous expression, overexpression, and knockdown of KCNJ10.

• Western Blot Analysis: Verify single band of expected molecular weight (~42-43 kDa) .

• Multiple Antibody Comparison: Test antibodies targeting different epitopes of KCNJ10 and compare staining patterns.

• Tissue Distribution Analysis: Compare antibody staining with known KCNJ10 expression patterns in brain, kidney, and inner ear.

What are the optimal conditions for KCNJ10 antibody use in Western blotting?

For optimal Western blot results with KCNJ10 antibodies:

Protocol Recommendations:

• Sample Preparation: Use lysis buffers containing protease inhibitors to prevent degradation

• Protein Loading: 20-50 μg total protein per lane

• Blocking Solution: 5% non-fat dry milk in TBST is recommended

• Primary Antibody Dilution: Typically 1:500 to 1:2000 (optimize for specific antibody)

• Expected Band Size: ~42-43 kDa

• Controls: Include positive control tissues (kidney, brain) and loading controls (β-actin)

Troubleshooting Tips:

• If multiple bands appear, optimize antibody concentration or blocking conditions

• For weak signals, increase protein loading or extend primary antibody incubation

• For high background, increase washing steps or adjust antibody dilution

What protocol modifications are needed for successful immunohistochemical/immunofluorescence detection of KCNJ10?

For effective IHC/IF detection of KCNJ10:

Recommended Protocol:

• Fixation: 4% paraformaldehyde is commonly used

• Antigen Retrieval: May be necessary for formalin-fixed, paraffin-embedded tissues

• Blocking: Use serum from secondary antibody species

• Primary Antibody Dilution: 1:100 for immunofluorescence labeling

• Detection Systems: DAB or fluorescent secondary antibodies

• Counterstaining: DAPI for nuclear visualization

Tissue-Specific Considerations:

• Brain: Focus on glial cell populations

• Kidney: Examine distal convoluted tubules

• Inner Ear: Target stria vascularis intermediate cells

For multiplex staining, as demonstrated with KCNJ10 and α1-Syntrophin, use primary antibodies from different host species (e.g., guinea pig anti-KCNJ10 and rabbit anti-α1-Syntrophin) to prevent cross-reactivity .

How can KCNJ10 antibodies be used to investigate EAST/SeSAME syndrome mutations?

EAST/SeSAME syndrome results from mutations in KCNJ10. Antibodies can be valuable tools in studying these mutations:

• Localization Studies: Compare subcellular localization of wild-type and mutant KCNJ10 proteins using immunofluorescence.

• Expression Analysis: Quantify expression levels of mutant proteins relative to wild-type using Western blotting.

• Heterologous Expression Systems: Following identification of mutations like those described (p.F75C, p.A167V, p.V91fs197X) , express mutants in systems like Xenopus oocytes and correlate functional analysis with antibody-based protein expression quantification.

• Co-expression Studies: Investigate effects on heteromeric channels by co-expressing KCNJ10 mutants with KCNJ16, as some mutations (e.g., p.A167V) show mild effects alone but severe effects in heteromeric channels .

• Animal Models: Use antibodies to analyze KCNJ10 expression in transgenic models expressing human mutations.

What approaches can be used for quantifying KCNJ10 expression in different experimental systems?

Multiple complementary methods can be employed for accurate KCNJ10 quantification:

• mRNA Quantification (qPCR):

  • Primers: Forward 5′-CTGAAAAGCTCAAGTTGGAGGA-3′, reverse 5′-GTAATCTGGAACATCGTATGGGTAG-3′

  • Reference gene: β-actin (ACTB)

  • Analysis: Comparative Ct method expressing results as the ratio of KCNJ10 to reference gene

• Protein Quantification (Western Blotting):

  • Detect KCNJ10 at ~42-43 kDa

  • Normalize to housekeeping proteins

  • Perform densitometric analysis of band intensity

• Immunohistochemical Quantification:

  • Standardize image acquisition settings

  • Quantify cell numbers, staining intensity, or area measurements

  • Use software tools like ImageJ or CellProfiler

• Transgenic Reporter Models:

  • Tg(Kcnj10-ZsGreen) mice express ZsGreen under the Kcnj10 promoter

  • Allow direct visualization and quantification of cells expressing KCNJ10

  • Enable live and fixed tissue analysis

How can I use KCNJ10 antibodies in co-immunoprecipitation to study protein-protein interactions?

For successful co-immunoprecipitation (Co-IP) of KCNJ10 and interacting partners:

Protocol Considerations:

• Sample Preparation: Use mild non-ionic detergents (0.5-1% Triton X-100) to preserve protein-protein interactions while effectively solubilizing membrane proteins.

• Antibody Selection: Choose antibodies targeting regions not involved in protein interactions; both polyclonal and monoclonal antibodies can be effective, though each offers different advantages.

• Controls: Include IgG controls, input controls, and reciprocal IPs. For highest stringency, include samples from Kcnj10 knockout tissues.

• Potential Interacting Partners:

  • KCNJ16: Forms heteromeric channels with KCNJ10, critical for function

  • α1-Syntrophin: Shows partial co-localization with KCNJ10 in blood vessels

  • Other scaffolding and regulatory proteins

• Technical Challenges: Membrane protein solubilization may require optimization of detergent type and concentration; consider chemical cross-linking to stabilize transient interactions.

How can KCNJ10 antibodies be used with advanced imaging techniques?

KCNJ10 research is increasingly employing sophisticated imaging approaches:

• Super-resolution Microscopy: Techniques like STORM or STED can reveal nanoscale organization of KCNJ10 channels relative to interacting proteins and cellular structures.

• Multiplex Imaging: As demonstrated with KCNJ10 and α1-Syntrophin co-staining in rat fornix, multiplex approaches can reveal complex spatial relationships between KCNJ10 and other cellular components .

• 3D Tissue Imaging: Tissue clearing methods combined with KCNJ10 immunolabeling enable whole-organ analysis of channel distribution.

• Live Imaging with Reporter Models: Transgenic Tg(Kcnj10-ZsGreen) mice allow visualization of KCNJ10-expressing cells in living tissues .

What methods can be used to correlate KCNJ10 localization with channel function?

To connect KCNJ10 distribution with its physiological roles:

• Electrophysiology with Immunocytochemistry:

  • Patch-clamp recordings from identified cells followed by immunostaining

  • Correlation of channel density with current amplitudes

• PIP2 Sensitivity Testing:

  • Assess channel interactions with phospholipids using poly-lysine clustering of PIP2

  • Use the time course of inhibition to estimate KCNJ10/PIP2 interaction strength

• pH Sensitivity Analysis:

  • Examine channel response to pH changes using inside-out patch configurations

  • Apply different pH solutions to the intracellular side of membranes via multibarrel perfusion systems

• Pharmacological Modulation:

  • Test specific blockade by barium and cesium

  • Compare wild-type and mutant channel responses to modulators

How can KCNJ10 antibodies facilitate research in developmental biology and disease models?

KCNJ10 antibodies enable investigation of expression changes in development and pathological conditions:

• Developmental Studies:

  • Track KCNJ10 expression throughout embryonic and postnatal development

  • Correlate expression changes with functional maturation of tissues

  • The transgenic Tg(Kcnj10-ZsGreen) mouse model allows visualization of developmental expression patterns

• Disease Models:

  • EAST/SeSAME Syndrome: Compare localization and expression of wild-type and mutant KCNJ10

  • Epilepsy: Examine alterations in KCNJ10 expression in glial cells

  • Hearing Loss: Investigate KCNJ10 in stria vascularis dysfunction

  • Renal Disorders: Study expression changes in salt-wasting tubulopathies

• Therapeutic Development:

  • Screen compounds that might restore function of mutant KCNJ10 channels

  • Monitor effects of treatments on KCNJ10 expression and localization

  • Use antibodies in high-throughput screening assays