KCNN1 Antibody

What is KCNN1 and why are antibodies against it important for research?

KCNN1 is a small conductance calcium-activated potassium channel (also known as KCa2.1 or SK1) that is voltage-insensitive and activated by intracellular Ca²⁺ in the submicromolar range. It has a similar topology to voltage-dependent K⁺ channels with six transmembrane domains and intracellular N- and C-termini .

Antibodies against KCNN1 are crucial research tools because:

• They enable visualization of KCNN1 expression patterns in different tissues

• They help determine subcellular localization of the channel

• They can be used to study KCNN1's role in various disease models

• They facilitate investigation of KCNN1's interactions with other proteins

Validated KCNN1 antibodies have been used in various applications including Western blot, immunohistochemistry, and immunofluorescence studies in tissues like brain cortex, hippocampus, and kidney sections .

What validation methods should be used to confirm KCNN1 antibody specificity?

For rigorous validation of KCNN1 antibodies, researchers should employ multiple complementary approaches:

• Peptide blocking experiments: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining, as demonstrated in rat brain sections where pre-incubation with KCNN1 blocking peptide suppressed immunoreactivity in cortical neurons and hippocampal pyramidal cells .

• Western blot analysis:

  • Use positive control tissues known to express KCNN1 (e.g., rat brain cortex)

  • Include negative controls where KCNN1 is absent or knocked down

  • Verify single band of expected molecular weight

  • Compare with siRNA knockdown samples to confirm specificity

• Immunohistochemical controls:

  • Compare staining patterns with known KCNN1 expression profiles

  • Include secondary antibody-only controls

  • Use knockout/knockdown tissues when available

• Cross-reactivity testing: Test against related potassium channels (KCa2.2/SK2, KCa2.3/SK3) to confirm specificity to KCNN1 .

How can KCNN1 antibodies be used for subcellular localization studies?

For accurate subcellular localization studies of KCNN1:

• Tissue preparation techniques:

  • For brain tissue: Use perfusion-fixed frozen sections for optimal preservation of cellular architecture

  • Use appropriate antigen retrieval methods if necessary

• Immunofluorescence methodology:

  • Combine KCNN1 antibodies with markers for specific subcellular compartments

  • In rat brain cortex, KCNN1 immunoreactivity appears in outlines of cortical neurons

  • In hippocampal CA1 region, KCNN1 staining is observed in the pyramidal cell layer and apical dendrites

• High-resolution imaging techniques:

  • Confocal microscopy is essential for precise localization

  • Use nuclear counterstains (e.g., DAPI) for reference

  • Consider super-resolution microscopy for nanoscale localization of channel clusters

• Co-localization studies:

  • In co-expression studies with fluorescent-tagged proteins (e.g., KCNN1-EGFP), examine localization patterns in relation to other cellular proteins or structures

In neurons, KCNN1 shows characteristic distribution patterns, with staining often excluded from the nucleus (or at minimum from the nucleolus) and extending into neuronal processes .

What are the optimal protocols for using KCNN1 antibodies in Western blotting?

For optimal Western blotting with KCNN1 antibodies:

Sample preparation:

• Use appropriate extraction buffers containing protease inhibitors

• For brain tissues, cortical samples have shown consistent KCNN1 expression

• Include positive controls (brain cortex) and negative controls

Protocol recommendations:

• Antibody dilution: 1:200 has been successfully used for rat brain cortex samples

• Blocking: 5% non-fat dry milk or BSA in TBST (experiment to determine optimal)

• Verification: Always confirm specificity by pre-incubating with KCNN1 blocking peptide

• Detection methods: Both chemiluminescence and fluorescent secondary antibodies have been successfully used

Troubleshooting:

• Multiple bands may indicate different isoforms or post-translational modifications

• High background may be reduced by further antibody dilution or more stringent washing

• For faint signals, extend exposure time or consider using signal enhancing systems

How can KCNN1 antibodies be effectively used in immunohistochemistry and immunofluorescence applications?

For immunohistochemistry/immunofluorescence:

Tissue preparation:

• For brain studies: Perfusion fixation with 4% paraformaldehyde followed by either paraffin embedding or cryoprotection and freezing

• For breast cancer tissues: Deparaffinization with xylene followed by rehydration in graded ethanol, antigen retrieval in citric acid buffer (pH 6.0)

Protocol steps:

• Antigen retrieval: Microwave heating in citric acid buffer (pH 6.0) for tissue microarrays

• Permeabilization: 0.1-1% Triton X-100 in PBS for 10-15 minutes

• Blocking: 5-10% normal serum in PBS-T for 30-60 minutes

• Primary antibody: 1:200 dilution for KCNN1 antibody (APC-039), incubated overnight at 4°C

• Detection: For fluorescence, goat anti-rabbit-AlexaFluor-488 has been successfully used

Application examples:

• In rat parietal cortex: KCNN1 immunoreactivity appears in outlines of cortical neurons

• In rat hippocampus: Staining is observed in pyramidal cell layer and apical dendrites of CA1 region

• In breast cancer tissues: IHC with KCNN1 antibodies has been used to compare expression between cancer and paracancer tissues

What techniques enable reliable KCNN1 knockdown for antibody validation and functional studies?

Several approaches have proven effective for KCNN1 knockdown:

siRNA-based knockdown:

• TC-71 cells transfected with KCNN1 siRNA (ON-TARGETplus siRNA, Dharmacon) using Lipofectamine RNAiMAX Reagent achieved 75% decrease in KCNN1 expression compared to scrambled siRNA controls

• Microinjection of KCNN1-specific siRNA into dorsal root ganglia achieved 54% decrease in mRNA and 63% decrease in protein levels compared to scrambled siRNA controls

Protocol considerations:

• Transfection optimization: Cell density of approximately 70% confluence is optimal for transfection

• Verification methods:

  • RT-qPCR 24h post-transfection confirms knockdown at mRNA level

  • Western blot 48-72h post-transfection confirms protein reduction

• Controls: Always include scrambled siRNA as control

• Specificity verification: Confirm that related potassium channels (e.g., K2p1.1, Kv1.2) remain unaffected to demonstrate specificity

Functional validation:

• In dorsal root ganglion studies, KCNN1 knockdown significantly increased neuronal excitability, confirming functional consequences of reduced KCNN1 levels

• The specificity of knockdown can be validated by rescue experiments using KCNN1 overexpression

How does KCNN1 expression and function differ across neurological disease models?

KCNN1 shows distinct expression patterns and functional impacts across different neurological conditions:

Neuropathic Pain Models:

• In chronic constriction injury (CCI) and spinal nerve ligation (SNL) models, KCNN1 is significantly downregulated in dorsal root ganglia (DRG)

• Temporal pattern: mRNA levels decreased by 58%, 50%, and 91% on days 3, 7, and 14 after CCI, respectively

• Cell specificity: KCNN1 is preferentially localized to small and medium neuronal bodies in DRGs, with about 87% of KCNN1-positive neurons labeled by IB4 (marker for nonpeptidergic nociceptor) and 56% labeled by CGRP (marker for peptidergic nociceptor)

Neurodegenerative Disease Models:

• In ALS models (G85R SOD1YFP and G93A SOD1 mice), overexpression of KCNN1 extends survival time

• In G85R SOD1YFP mice, KCNN1 overexpression extends median survival by up to 100%

• In A53T alpha-synuclein transgenic mice (Parkinson's model), KCNN1 overexpression extends median time to endstage motor disease by >100%

• Mechanism: Overexpressed KCNN1 induces protective stress responses including ER stress response, mitochondrial stress response, and integrated stress response

Ewing Sarcoma:

• KCNN1 is highly expressed in Ewing sarcoma due to direct regulation by the main oncogene

• Despite high expression, the channel is non-functional in these cells

• This lack of K+ conductance makes Ewing sarcoma cells vulnerable to hypoosmotic stress

Functional implications:

• In neuropathic pain, KCNN1 downregulation increases neuronal excitability

• In neurodegenerative diseases, KCNN1 overexpression is protective through stress response pathways

• Different disease contexts may require different targeting strategies (restoration vs. inhibition)

What role does KCNN1 play in cancer and how can antibodies help investigate its mechanisms?

KCNN1 has emerging roles in cancer biology, particularly in breast cancer:

Expression in cancer:

• KCNN1 is overexpressed in breast cancer tissues and cells compared to normal tissue

• Immunohistochemical staining using tissue microarrays containing 140 breast cancer tissue samples and 90 paracancer tissue samples has confirmed this overexpression

Functional roles:

• KCNN1 functions as an oncogene promoting breast cancer cell proliferation and metastasis

• Mechanistically, KCNN1 interacts with ERLIN2 and enhances the effect of ERLIN2 on Cyclin B1 stability

• KCNN1 overexpression promotes Cyclin B1 protein expression, enhances its stability, and promotes its K63-dependent ubiquitination

Research applications of antibodies:

• Protein-protein interaction studies: Immunoprecipitation with KCNN1 antibodies can identify binding partners like ERLIN2

• Co-localization analysis: Immunofluorescence using KCNN1 antibodies in combination with antibodies against interaction partners

• Expression correlation studies: IHC analysis of paired tissue samples has revealed positive expression correlations between KCNN1 and ERLIN2, KCNN1 and Cyclin B1, as well as ERLIN2 and Cyclin B1

Methodological approaches:

• Co-immunoprecipitation: Using KCNN1 antibodies to pull down protein complexes

• Fluorescent protein tagging: KCNN1-EGFP and ERLIN2-DSRED co-transfection in MCF7 cells allows visualization of co-localization using confocal microscopy

• Stability assays: Using proteasome inhibitors (MG132) at different time points to assess the effect of KCNN1 on protein stability of targets like Cyclin B1

How can KCNN1 antibodies help elucidate the channel's role in neuropathic pain?

KCNN1 antibodies have been instrumental in understanding the channel's role in neuropathic pain:

Expression analysis in pain models:

• Immunohistochemistry using KCNN1 antibodies shows diminished expression in dorsal root ganglia after nerve injury

• Temporal analysis reveals progressive reduction in KCNN1-positive neurons following chronic constriction injury (CCI) or spinal nerve ligation (SNL)

Neuronal subtype characterization:

• Co-staining with KCNN1 antibodies and neuronal markers (IB4, CGRP) shows preferential expression in nociceptors

• About 87% of KCNN1-positive DRG neurons are labeled by IB4 (marker for nonpeptidergic nociceptor)

• Approximately 56% of KCNN1-positive DRG neurons are labeled by CGRP (marker for peptidergic nociceptor)

Functional correlation studies:

• Combining KCNN1 immunostaining with electrophysiological recordings to correlate expression with altered neuronal excitability

• After nerve injury, downregulation of KCNN1 correlates with increased neuronal hyperexcitability

Therapeutic target validation:

• KCNN1 restoration through viral vector delivery (AAV9-KCNN1) microinjected into DRGs leads to increased KCNN1 protein levels

• Immunohistochemistry confirms successful restoration of KCNN1 expression

• This restoration correlates with alleviation of pain behaviors, suggesting therapeutic potential

How can KCNN1 antibodies be used to investigate the channel's involvement in novel protective mechanisms against neurodegeneration?

Recent research has revealed unexpected protective roles for KCNN1 in neurodegenerative diseases:

Research approaches using antibodies:

• Expression analysis in disease progression:

  • Immunostaining with KCNN1 antibodies in spinal cord cross-sections of ALS model mice (G85R SOD1YFP mice)

  • KCNN1 antibody staining reveals diffuse cytoplasmic localization in motor neurons with nuclear exclusion

  • Quantitative analysis shows ~5-10 fold elevation of intensity in transgenic compared to non-transgenic spinal cord

• Co-localization with pathological proteins:

  • Combined immunostaining for KCNN1 and YFP fluorescence (representing G85R SOD1YFP protein)

  • KCNN1 shows patchy cytoplasmic pattern while SOD1YFP shows more uniform distribution

  • Merged images reveal considerable regional overlap without precise co-localization

• Viral vector delivery assessment:

  • KCNN1 antibody immunostaining confirms successful AAV-mediated overexpression of KCNN1 in motor neurons

  • P0 injection of AAV9-Kcnn1 achieves virtually 100% motor neuron transduction of ChAT-positive large neurons in ventral horn

• Stress response pathway investigation:

  • Antibodies against KCNN1 and stress response markers reveal that KCNN1 overexpression induces protective stress responses

  • These include ER stress, mitochondrial stress, and integrated stress responses

  • These stress responses appear to protect against SOD1 and alpha-synuclein pathologies

Mechanistic insights:

• KCNN1 appears to be channel-inactive in this context due to absence of Kcnn2 with which to co-assemble

• The diffuse cytoplasmic localization (rather than membrane localization) supports this non-canonical function

• The non-assembled, potentially misfolded state of overexpressed KCNN1 targeted to the ER may explain the induced stress responses that are protective

What are the methodological considerations for developing monoclonal antibodies against KCNN1?

Development of monoclonal antibodies against KCNN1 requires careful consideration:

Epitope selection strategies:

• Target unique regions: Focus on regions with low homology to related channels (KCNN2/SK2, KCNN3/SK3)

• Functional domains: Consider targeting extracellular loops or specific functional domains

• Species conservation: For cross-species applications, target conserved epitopes

  • Examples: Peptide (C)DRPGSGKPPTVSHRLGHRR from amino acid residues 75-93 of rat KCNN1 (N-terminal region) has been successfully used

Production methods:

• Hybridoma technology: Enables selection of high-affinity clones

  • Screening must include functional validation beyond binding assays

  • Out of 40 hybridoma clones, only a small number may produce functional antibodies with the desired specificity

• Purification and characterization:

  • Surface plasmon resonance (SPR) for determining binding kinetics and affinity constants

  • High-resolution conformational epitope mapping to identify exact binding sites

Validation strategies:

• Binding assays: ELISA to confirm antibody binding to target epitope

• Specificity testing: Western blot against tissues known to express KCNN1

• Functional validation: Patch-clamp experiments in cells expressing KCNN1 to assess if antibodies affect channel function

• Cross-reactivity testing: Test against related channels to ensure specificity

Application optimization:

• Determine optimal working dilutions: Typically 1:100 to 1:200 for immunostaining

• Test fixation conditions: Perfusion-fixed vs. paraformaldehyde-fixed preparations

• Optimize immunostaining protocols: Antigen retrieval, blocking, and incubation conditions

What techniques can be used to investigate KCNN1 interactions with other proteins and their functional consequences?

Investigating KCNN1 protein interactions requires sophisticated techniques:

Protein interaction identification methods:

• Co-immunoprecipitation (Co-IP) using KCNN1 antibodies:

  • Pull-down of KCNN1 followed by probing for suspected interaction partners

  • Reverse Co-IP using antibodies against potential binding partners

  • Examples: KCNN1 interaction with ERLIN2 has been demonstrated this way

• Proximity ligation assay (PLA):

  • Detects protein-protein interactions in situ with single-molecule resolution

  • Requires specific antibodies for both KCNN1 and interaction partner

  • Signal amplification enables detection of even transient interactions

• Fluorescence resonance energy transfer (FRET):

  • KCNN1-EGFP and partner-DSRED co-transfection allows visualization of co-localization

  • MCF7 cells co-transfected with KCNN1-EGFP and ERLIN2-DSRED constructs can be visualized using confocal microscopy

Functional consequence assessment:

• Protein stability assays:

  • Proteasome inhibitor (MG132) time course experiments

  • KCNN1's effect on Cyclin B1 stability has been demonstrated using this approach

• Ubiquitination assays:

  • KCNN1 has been shown to promote K63-dependent ubiquitination of Cyclin B1

  • This requires immunoprecipitation followed by ubiquitin-specific antibody detection

• siRNA-based validation:

  • Knockdown of interaction partners (e.g., ERLIN2) can partially restore effects induced by KCNN1 overexpression

  • This approach confirms functional relationship between the proteins

Correlation in clinical samples:

• Paired tissue samples can be analyzed by IHC to assess correlation between KCNN1 and interaction partners

• Positive expression correlations between KCNN1 and ERLIN2, KCNN1 and Cyclin B1, as well as ERLIN2 and Cyclin B1 have been demonstrated in breast cancer samples

How should researchers interpret discrepancies between KCNN1 mRNA expression and protein detection?

Discrepancies between KCNN1 mRNA and protein levels are common and should be systematically analyzed:

Common patterns of discrepancy:

• High mRNA with low/undetectable protein:

  • In Ewing sarcoma, high KCNN1 mRNA expression (driven by the main oncogene) doesn't translate to functional channel protein

  • Despite verification in samples from >200 patients, the channel is not functional and does not modulate cell behavior

• Dynamic temporal changes:

  • After nerve injury (CCI model), KCNN1 mRNA levels decreased by 58%, 50%, and 91% on days 3, 7, and 14 respectively

  • Protein levels follow similar pattern but with different magnitudes or timing

Methodological considerations:

• Transcript variant detection:

  • RT-PCR with exon-specific primers can detect different KCNN1 variants

  • Previously unknown KCNN1 variants have been identified in some contexts

• Protein detection optimization:

  • Different antibodies may recognize different epitopes or isoforms

  • Western blot conditions may need optimization for different tissues

  • Consider detergent solubility - membrane proteins may require special extraction methods

• Quantification approaches:

  • For mRNA: RT-qPCR with validated reference genes

  • For protein: Densitometry of Western blots with appropriate loading controls

Biological explanations:

• Post-transcriptional regulation: miRNAs or RNA-binding proteins may affect translation

• Protein stability issues: Rapid degradation of KCNN1 protein

• Subcellular trafficking defects: Protein may be synthesized but not properly localized

• Context-dependent expression: Different cellular environments affect protein expression

How can researchers determine if KCNN1 is functionally active versus merely expressed in their experimental system?

Distinguishing between KCNN1 expression and functional activity requires specific approaches:

Functional assessment techniques:

• Electrophysiological methods:

  • Patch-clamp recordings to measure KCNN1-mediated currents

  • Characteristic small conductance calcium-activated K+ currents indicate functional channels

  • In Ewing sarcoma, despite high expression, absence of KCNN1-carried K+ current indicates non-functional channels

• Calcium sensitivity testing:

  • KCNN1 is activated by intracellular Ca2+ in the submicromolar range

  • Manipulate intracellular calcium levels while monitoring K+ currents

  • Specific KCNN channel blockers can confirm current identity

• Regulatory volume decrease (RVD) assays:

  • Functional KCNN1 contributes to cell volume regulation

  • Compare RVD in cells with normal versus altered KCNN1 expression

  • KCNN1 siRNA-treated cells show altered RVD responses

• Pharmacological manipulation:

  • Apply specific KCNN channel modulators (activators or inhibitors)

  • Observe cellular responses or electrophysiological changes

  • Lack of response suggests non-functional channels

Insights from disease models:

• Context-dependent functionality:

  • In Ewing sarcoma: KCNN1 is highly expressed but non-functional

  • This creates vulnerability to hypoosmotic stress that can be therapeutically exploited

• Non-canonical functions:

  • In ALS models: Overexpressed KCNN1 is diffusely cytoplasmic rather than membrane-localized

  • It exerts protective effects through stress responses rather than channel activity

  • KCNN1 may be channel-inactive due to absence of Kcnn2 with which to co-assemble

• Assembly requirements:

  • Functional KCNN1 channels may require co-assembly with other subunits

  • Expression of KCNN1 alone may result in non-functional protein

  • Investigate expression of potential partner subunits in your system

Table 1: Common Applications of KCNN1 Antibodies in Research