Recombinant Human FXYD domain-containing ion transport regulator 7 (FXYD7)

What is FXYD7 and how does it relate to the larger FXYD protein family?

FXYD7 is a member of the FXYD family of small membrane proteins that share a 35-amino acid signature sequence domain beginning with the sequence PFXYD and containing 7 invariant and 6 highly conserved amino acids. The FXYD family was defined following searches for homologues of the Na,K-ATPase γ subunit, CHIF, and phospholemman in EST and gene data banks . FXYD7 is uniquely brain-specific among this family and functions as a regulator of the Na,K-ATPase . Like other FXYD family members, it has a type I membrane protein structure with an extracellular N-terminus and a cytoplasmic C-terminus . The human FXYD7 gene is located on chromosome 19q13.12 .

How should researchers approach the storage and handling of recombinant FXYD7?

Recombinant FXYD7 antibodies should be stored at -20°C in PBS with 50% glycerol and 0.1% sodium azide at pH 7.3 . For experimental applications, it's important to maintain the protein in wet ice during handling and preparation . When planning experiments, researchers should consider that the typical stock concentration for commercially available recombinant FXYD7 antibodies is approximately 0.32 mg/mL . The antibodies demonstrate reactivity across human, mouse, and rat samples, making them versatile for comparative studies .

What is the tissue distribution pattern of FXYD7 and how can it be accurately determined?

FXYD7 exhibits a strict brain-specific expression pattern. This can be definitively demonstrated through northern and western blot analyses across multiple tissues. Research has shown a strong signal corresponding to a 0.7 kb mRNA exclusively in brain tissue, with no detection in other organs . Within the brain, FXYD7 mRNA and protein levels vary by region, with highest expression in cerebral structures and lowest in the hypothalamus . To accurately determine tissue distribution, researchers should employ multiple complementary techniques:

• Northern blot analysis for mRNA detection

• Western blot analysis for protein detection

• Immunohistochemistry for spatial localization

• Q-rtPCR for quantitative measurement

These methods should be performed with appropriate controls to ensure specificity, particularly important for distinguishing FXYD7 from other FXYD family members .

What post-translational modifications occur in FXYD7 and how do they affect protein function?

FXYD7 undergoes specific post-translational modifications, primarily on threonine residues in its N-terminal domain. These modifications occur specifically on Thr3, Thr5, and Thr9 residues . The modifications are most likely O-glycosylations, as suggested by partial sensitivity to neuraminidase and O-glycosidase treatments . These modifications are added post-translationally rather than co-translationally, as evidenced by pulse-chase experiments in Xenopus oocytes showing the appearance of modified forms after a 24-hour pulse .

The functional significance of these modifications includes:

• Protein stabilization - N-terminally modified FXYD7 shows greater stability compared to unmodified forms

• Proper cellular trafficking - modifications may influence intracellular routing or membrane targeting

• Potentially modulating protein-protein interactions

Importantly, research has shown that these modifications are not required for the functional effect of FXYD7 on K+ affinity of Na,K-ATPase, nor do they influence the Na+ affinity of Na,K-ATPase . To study these modifications, researchers should employ site-directed mutagenesis of threonine residues, glycosidase treatments, and metabolic labeling approaches.

How can researchers distinguish between different post-translationally modified forms of FXYD7?

Different post-translationally modified forms of FXYD7 can be distinguished through several complementary techniques:

• SDS-PAGE mobility analysis: Modified FXYD7 appears as bands of approximately 18-19 kDa compared to the 14 kDa core protein .

• Glycosidase treatments: Sequential treatment with neuraminidase and O-glycosidase can be used to assess the presence and extent of O-glycosylation.

• Mass spectrometry: For precise characterization of modifications, liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides the most definitive identification of specific modifications on individual amino acid residues.

• Site-directed mutagenesis: Mutation of individual threonine residues (Thr3, Thr5, Thr9) to alanine and expression in cellular systems can confirm the specific sites of modification.

When expressing FXYD7 in experimental systems, researchers should note that native brain FXYD7 appears primarily as an 18 kDa band, which corresponds to the most prominent form seen when expressing FXYD7 in Xenopus oocytes . This suggests that the oocyte expression system processes FXYD7 similarly to native brain tissue, making it a suitable model for studying FXYD7 modifications.

Which Na,K-ATPase isozymes does FXYD7 interact with and how can these interactions be experimentally demonstrated?

To experimentally demonstrate these interactions, researchers should employ:

• Co-immunoprecipitation: Using antibodies against either FXYD7 or specific Na,K-ATPase α subunits to pull down protein complexes, followed by western blotting to detect associated proteins.

• Co-expression in heterologous systems: Expression of FXYD7 with various Na,K-ATPase isozymes in Xenopus oocytes, followed by metabolic labeling and immunoprecipitation under non-denaturing conditions.

• FRET or BiFC analysis: For live-cell visualization of protein-protein interactions.

• Cross-linking studies: To stabilize transient interactions prior to isolation.

Key experimental controls should include:

• Immunoprecipitation with non-specific IgG

• Expression of individual proteins alone

• Competition with excess untagged protein

The detection of stable associations even after prolonged chase periods in metabolic labeling experiments indicates that these interactions are specific and physiologically relevant .

How does FXYD7 modify the functional properties of Na,K-ATPase?

FXYD7 specifically modulates the K+ transport properties of Na,K-ATPase without affecting Na+ kinetics. The key functional effects include:

• Decreased apparent K+ affinity: FXYD7 decreases the apparent affinity for extracellular K+ of Na,K-ATPase α1–β1 complexes approximately 2-fold over a wide potential range . This effect is observed both in the presence and absence of external Na+, suggesting a modification of the intrinsic affinity of the external K+-binding site rather than an indirect effect through Na+ binding.

• Isozyme-specific modulation: FXYD7 modifies the K+ affinity of α1–β1 and α2–β1 isozymes but not α3–β1 isozymes, despite its ability to physically associate with all three isozymes .

• Enhanced Na+ inhibition: In FXYD7-associated α1–β1 complexes, Na,K-ATPase pump activity is inhibited more strongly by external Na+ at high negative membrane potentials, suggesting effects on Na+ translocation and release .

These functional effects can be quantitatively demonstrated through electrophysiological measurements in expression systems (e.g., Xenopus oocytes) using techniques such as:

• Two-electrode voltage clamp to measure pump currents

• K+ activation curves at various membrane potentials

• Measurement of K1/2K+ values in the presence or absence of Na+

The following table summarizes the effect of FXYD7 on K1/2K+ values for different Na,K-ATPase isozymes:

What is the proposed physiological role of FXYD7 in neuronal K+ homeostasis?

FXYD7 plays a crucial role in adapting Na,K-ATPase function for optimal K+ homeostasis during neuronal activity. The current evidence suggests several physiological functions:

• Tailoring K+ uptake to activity levels: By decreasing the apparent K+ affinity of α1–β1 Na,K-ATPase complexes, FXYD7 ensures that under resting conditions (with [K+]o ~4 mM), these pumps do not operate at maximum capacity . This creates a functional reserve that can be engaged during periods of increased neuronal activity when extracellular K+ concentrations rise.

• Preventing excessive K+ undershoot: After periods of intense neuronal activity, FXYD7-modified pumps help prevent excessive K+ undershoot that could lead to hyperpolarization and reduced excitability .

• Cell-type specific regulation: The selective association of FXYD7 with α1–β1 complexes in the brain provides a mechanism for cell-type specific regulation of K+ transport properties, complementing the differential expression of Na,K-ATPase isozymes .

• Adaptation to brain-specific ion transport needs: The brain-specific expression of FXYD7 reflects its specialized role in meeting the unique ion transport requirements of neural tissue, where rapid changes in extracellular K+ concentration accompany neuronal activity .

Experimental evidence supporting these roles comes from electrophysiological studies showing that FXYD7-associated α1–β1 Na,K-ATPase has a K1/2K+ of approximately 2 mM, compared to ~1 mM for unassociated pumps . This shift means that in the presence of FXYD7, the pump retains capacity to increase its activity as [K+]o rises from 4 mM to higher levels during neuronal activity.

How do astrocytes and neurons differ in their FXYD7 expression and Na,K-ATPase characteristics, and what are the implications for K+ clearance?

Astrocytes and neurons exhibit significant differences in their FXYD7 expression patterns and Na,K-ATPase characteristics, which influence their respective roles in K+ homeostasis:

Cellular differences in Na,K-ATPase subunit composition:

• Neurons express α1, α3, and β1 subunits

• Astrocytes predominantly express α1, α2, and β1 subunits

Differences in FXYD7 association and Na,K-ATPase function:
Based on ouabain binding kinetics and FXYD7 expression studies, the following patterns emerge:

The Bmax values for these subunits provide insight into their relative abundance:

• α2 and α3 subunits: 15-30 pmol/mg protein

• α1 subunit in neurons: low

• α1 subunit in astrocytes: very high (645 pmol/mg protein)

Implications for K+ clearance:

• The high density but slow turnover of FXYD7-associated α1 pumps in astrocytes provides a large-capacity system for long-term K+ homeostasis

• The higher-turnover α2 pumps in astrocytes likely contribute to rapid K+ clearance following neuronal activity

• Neuronal α3 pumps, unaffected by FXYD7, maintain consistent function even during significant changes in extracellular K+

• The differential association of FXYD7 creates a coordinated system where K+ uptake rates in different cell types are matched to their physiological roles

This arrangement ensures that during periods of intense neuronal activity, the resultant increases in extracellular K+ can be efficiently managed through a combination of rapid uptake systems and longer-term homeostatic mechanisms.

What are the most effective methods for studying FXYD7 expression and localization in brain tissue?

Multiple complementary techniques should be employed to comprehensively study FXYD7 expression and localization:

1. mRNA Detection:

• Northern blot analysis: Effective for tissue-level expression, showing 0.7 kb FXYD7 mRNA exclusively in brain tissue

• In situ hybridization: For cellular and regional localization of FXYD7 mRNA within brain sections

• Quantitative real-time PCR (Q-rtPCR): For precise quantification of expression levels across brain regions, using protocols similar to those described for other FXYD family members

2. Protein Detection:

• Western blot analysis: Using specific antibodies for FXYD7 (such as HPA026916 or 11465-1-AP), with expected bands at ~18 kDa in brain lysates

• Immunoprecipitation: Useful for studying protein-protein interactions, especially with Na,K-ATPase subunits

3. Cellular Localization:

• Immunofluorescence microscopy: For precise cellular and subcellular localization

• Double immunofluorescence: Essential for determining cell-type specificity, using established markers:

  • Neuronal markers: synaptophysin, NeuN

  • Glial markers: GFAP (for astrocytes), Iba1 (for microglia)

• Confocal microscopy: For high-resolution co-localization studies

Methodological considerations:

• Use multiple antibodies targeting different epitopes to confirm specificity

• Include appropriate controls such as peptide competition assays

• For recombinant expression studies, Xenopus oocytes provide a system where FXYD7 processing occurs similarly to native brain tissue

• When studying regional distribution, systematic sampling across all major brain regions should be performed (cortex, cerebellum, hippocampus, striatum, etc.)

What experimental approaches are most suitable for investigating the functional effects of FXYD7 on Na,K-ATPase activity?

Several complementary approaches can effectively characterize the functional effects of FXYD7 on Na,K-ATPase:

1. Heterologous Expression Systems:

• Xenopus oocyte expression: The gold standard system for studying FXYD7-Na,K-ATPase interactions, allowing co-expression of specific Na,K-ATPase isozymes with FXYD7

• Mammalian cell expression systems: Complementary to oocytes, providing a more physiological environment

2. Electrophysiological Measurements:

• Two-electrode voltage clamp: For direct measurement of Na,K-ATPase pump currents in Xenopus oocytes

• Patch-clamp techniques: For measurements in mammalian cells

• K+ activation curves: Determination of K1/2K+ values at various membrane potentials

• Na+ inhibition studies: Assessing the effect of external Na+ on pump function

3. Biochemical Assays:

• Ouabain binding kinetics: For quantifying the number of pump sites (Bmax) and binding affinity (KD)

• 86Rb+ uptake assays: For direct measurement of K+ transport rates

• ATPase activity assays: Measuring inorganic phosphate production

4. Mutational Analysis:

• Site-directed mutagenesis: Of key residues in FXYD7 or Na,K-ATPase subunits

• Truncation mutants: To identify domains critical for interaction or function

• Chimeric constructs: Between different FXYD family members

5. Structural Studies:

• Cross-linking studies: To capture and analyze protein-protein interactions

• FRET/BRET: For studying dynamics of interactions in living cells

Experimental design considerations:

• Include appropriate controls (empty vector, non-interacting FXYD family members)

• Perform dose-response studies with varying FXYD7:Na,K-ATPase ratios

• Consider the effects of membrane potential, ion concentrations, and pH

• For in vivo relevance, validate key findings in primary neuronal or glial cultures

A particularly informative experimental paradigm combines electrophysiological measurements with biochemical quantification, as demonstrated in studies measuring both ouabain binding kinetics and functional K+ transport properties .

How might genetic manipulation of FXYD7 expression affect neuronal excitability and seizure susceptibility?

Given FXYD7's role in regulating K+ homeostasis, genetic manipulation of its expression could have significant implications for neuronal excitability:

Predicted effects of FXYD7 knockout:

• Enhanced K+ affinity of Na,K-ATPase α1–β1 complexes

• More rapid K+ clearance following neuronal activity

• Potential K+ undershoot, leading to hyperpolarization

• Reduced neuronal excitability, particularly following periods of intense activity

• Possible resistance to seizure induction due to enhanced K+ clearance

Predicted effects of FXYD7 overexpression:

• Further decreased K+ affinity of Na,K-ATPase

• Slower K+ clearance following neuronal activity

• Prolonged periods of elevated extracellular K+

• Increased neuronal excitability

• Potentially increased susceptibility to seizures or spontaneous seizure activity

Methodological approaches for these studies:

• Genetic models:

  • Conditional knockout mice (preferably with neuron-specific or astrocyte-specific targeting)

  • Viral-mediated overexpression systems

  • CRISPR/Cas9-mediated gene editing in primary cultures

• Functional readouts:

  • Extracellular K+ measurements using K+-sensitive microelectrodes

  • Field potential recordings in brain slices to assess network excitability

  • Whole-cell patch-clamp recordings to assess individual neuronal properties

  • In vivo EEG recordings for seizure susceptibility

  • Behavioral testing for seizure threshold using chemical convulsants

• Mechanistic validation:

  • Pharmacological manipulation of Na,K-ATPase using low-dose ouabain

  • K+ supplementation experiments

  • Combined manipulation of FXYD7 and specific Na,K-ATPase isozymes

When designing these experiments, researchers should consider that complete knockout of FXYD7 may lead to compensatory changes in other FXYD family members or Na,K-ATPase subunits, potentially complicating interpretation of results.

How does FXYD7 compare functionally to other brain-expressed FXYD family members, and what are the implications for differential regulation of Na,K-ATPase in neural tissues?

While FXYD7 is the primary brain-specific FXYD family member, other FXYD proteins are also expressed in neural tissues, creating a complex regulatory landscape:

Comparative analysis of brain-expressed FXYD proteins:

Key functional differences and their implications:

• Isozyme selectivity:

  • FXYD7's preferential association with α1–β1 in brain (despite ability to interact with α2/α3–β1 in vitro) suggests highly regulated targeting mechanisms

  • This selectivity allows for cell-type and subcellular-specific modulation of Na,K-ATPase function

• Ion specificity of effects:

  • FXYD7 selectively modulates K+ but not Na+ kinetics

  • FXYD1 primarily affects Na+ affinity

  • This differential regulation allows for independent control of Na+ and K+ homeostasis

• Post-translational regulation:

  • FXYD7 is regulated through O-glycosylation, which affects protein stability but not function

  • FXYD1 is dynamically regulated through phosphorylation events

  • These differences suggest distinct mechanisms for acute versus chronic modulation

Methodological approaches for comparative studies:

• Co-expression systems: Systematically comparing effects of different FXYD proteins on the same Na,K-ATPase isozymes in Xenopus oocytes

• Domain swapping experiments: Creating chimeric FXYD proteins to identify domains responsible for specific functional effects

• Cell-type specific analyses: Using single-cell RNA-seq and proteomic approaches to map the expression of FXYD family members across neural cell types

• Functional readouts: Comparing effects on Na+ versus K+ kinetics, membrane potential, and excitability

Understanding these differences has important implications for both basic neurophysiology and potential therapeutic interventions targeting specific aspects of Na,K-ATPase function in neurological disorders.

What techniques are most effective for investigating the structure-function relationship of FXYD7 and its interaction with Na,K-ATPase?

Elucidating the structure-function relationship of FXYD7 requires integration of multiple advanced techniques:

1. Structural Biology Approaches:

• X-ray crystallography: Of FXYD7-Na,K-ATPase complexes, building on existing crystal structures of Na,K-ATPase

• Cryo-electron microscopy: For visualization of the complete complex in different conformational states

• NMR spectroscopy: Particularly useful for studying the dynamics of interaction

• Molecular dynamics simulations: To predict conformational changes and energetics of interaction

2. Biochemical Interface Mapping:

• Crosslinking coupled with mass spectrometry: To identify contact points between FXYD7 and Na,K-ATPase

• Hydrogen-deuterium exchange mass spectrometry: For identifying protein regions involved in binding

• Alanine scanning mutagenesis: Systematic mutation of residues to identify critical interaction sites

• Peptide competition assays: Using synthetic peptides corresponding to putative interaction domains

3. Real-time Interaction Analysis:

• Surface plasmon resonance (SPR): For measuring binding kinetics and affinity

• Isothermal titration calorimetry (ITC): For thermodynamic characterization of binding

• Microscale thermophoresis: For measuring interactions in solution with minimal sample requirements

• FRET/BRET assays: For studying interaction dynamics in living cells

4. Functional Correlation Studies:

• Structure-guided mutagenesis: Based on structural findings, introducing specific mutations to test functional hypotheses

• Chimeric constructs: Between different FXYD family members to identify functional domains

• Electrophysiological measurements: Correlating structural features with functional effects on Na,K-ATPase transport properties

Key regions/residues for investigation:

• The FXYD motif itself and conserved residues in the signature sequence

• The transmembrane domain, likely involved in interaction with Na,K-ATPase

• The N-terminal region containing O-glycosylation sites

• The short C-terminal cytoplasmic domain

When designing these experiments, researchers should consider that disrupting the FXYD7-Na,K-ATPase interaction might affect protein stability or trafficking as well as direct functional effects, necessitating careful control experiments to distinguish these possibilities.

What role might FXYD7 play in neurological disorders associated with disrupted ion homeostasis?

Given its specific role in regulating Na,K-ATPase function and K+ homeostasis in the brain, FXYD7 could be implicated in several neurological disorders:

Potential involvement in epilepsy:

• Disrupted K+ clearance is a known contributor to seizure susceptibility

• Altered FXYD7 expression or function could affect the threshold for seizure initiation or termination

• Investigation approaches:

  • Analysis of FXYD7 expression in epileptic tissue from animal models and human samples

  • Correlation of genetic variants in FXYD7 with epilepsy phenotypes

  • Electrophysiological assessment of K+ dynamics in FXYD7-manipulated models

Cerebral ischemia and stroke:

• Na,K-ATPase dysfunction contributes to excitotoxic neuronal death following ischemia

• FXYD7's effect on K+ affinity could influence cellular responses during energy depletion

• Research directions:

  • Temporal profiling of FXYD7 expression following experimental ischemia

  • Assessment of neuroprotective potential of FXYD7 modulation

  • Investigation of FXYD7's role in astrocyte swelling during cytotoxic edema

Neurodegenerative disorders:

• Disrupted ion homeostasis is implicated in Alzheimer's, Parkinson's, and ALS

• FXYD7 could influence neuronal vulnerability to excitotoxicity or oxidative stress

• Experimental approaches:

  • Analysis of FXYD7 expression in post-mortem tissue and animal models

  • Investigation of FXYD7 interaction with disease-associated proteins

  • Assessment of whether FXYD7 modulation affects disease progression

Methodological considerations for these studies:

• Use multiple complementary models (in vitro, ex vivo, in vivo)

• Employ cell-type specific approaches to distinguish neuronal versus glial contributions

• Consider potential compensatory mechanisms involving other FXYD family members

• Utilize advanced imaging techniques to visualize real-time changes in ion concentrations

These investigations could potentially identify FXYD7 as a novel therapeutic target for disorders characterized by disrupted ion homeostasis and neuronal hyperexcitability.

How can advanced microscopy and electrophysiological techniques be combined to study FXYD7's role in dynamic K+ regulation during neuronal activity?

Integrating advanced imaging with electrophysiological techniques provides powerful approaches for studying FXYD7's role in real-time K+ dynamics:

Multimodal imaging approaches:

• K+-sensitive fluorescent indicators:

  • Combining K+ indicators (e.g., IPG-4) with cell-specific markers

  • Using genetically encoded K+ sensors (GINKO) for cell-type specific expression

  • Correlating local K+ changes with Na,K-ATPase activity

• Na,K-ATPase activity reporters:

  • FRET-based sensors for conformational changes in Na,K-ATPase

  • Fluorescent voltage indicators to track electrogenic pump activity

  • pH-sensitive fluorescent proteins to monitor proton countertransport

• Advanced microscopy platforms:

  • Two-photon microscopy for deep tissue imaging in intact preparations

  • Super-resolution techniques (STED, PALM/STORM) for nanoscale localization

  • Light-sheet microscopy for rapid volumetric imaging

Integrated electrophysiological approaches:

• K+-sensitive microelectrodes:

  • Ion-selective microelectrodes for direct [K+]o measurement

  • Combination with whole-cell recording to correlate with cellular activity

  • Spatial mapping of K+ dynamics in brain slice preparations

• Advanced patch-clamp techniques:

  • Isolating Na,K-ATPase currents using specific voltage protocols

  • Simultaneous recording from neuron-astrocyte pairs

  • Perforated patch recordings for minimally disruptive long-term measurements

Experimental paradigms for integrating these approaches:

• Activity-dependent K+ dynamics:

  • Induce controlled neuronal activity (electrical stimulation, optogenetics)

  • Simultaneously monitor [K+]o and Na,K-ATPase activity

  • Compare responses in wild-type versus FXYD7-manipulated preparations

• Cell-type specific contributions:

  • Use conditional expression/knockout of FXYD7 in specific cell populations

  • Correlate cellular Na,K-ATPase activity with local K+ clearance

  • Assess the impact of FXYD7 manipulation on network activity

• Subcellular localization and function:

  • Examine whether FXYD7-associated Na,K-ATPase complexes show preferential localization

  • Investigate possible co-localization with K+ channels or glutamate transporters

  • Determine if FXYD7 regulates functional coupling between transporters