Recombinant Human Transmembrane protein C15orf27 (C15orf27)

What is C15orf27/TMEM266 and where is it primarily expressed?

C15orf27, now officially known as TMEM266 (transmembrane protein 266), is a voltage-sensor protein present on the post-synaptic side of glutamatergic mossy fibers and granule cells in the cerebellum. Despite containing a voltage-sensor segment similar to those found in voltage-gated ion channels, it does not form a functional ion channel, and its precise physiological role remains under investigation .

The protein contains distinct domains including the S1-S4 voltage-sensing domain that shares sequence similarity with Hv1 (voltage-gated proton channel). It undergoes both rapid and slow structural rearrangements in response to changes in membrane voltage and contains a zinc-binding site that can regulate the slow conformational transition .

What are the key protein aliases and gene identifiers for human C15orf27/TMEM266?

The human C15orf27/TMEM266 protein is known by several aliases in scientific literature and databases:

This information is crucial for database searches and literature reviews when conducting research on this protein .

What expression systems are commonly used for producing recombinant C15orf27/TMEM266?

Recombinant human C15orf27/TMEM266 protein can be produced using various expression systems, with HEK293T cells being one of the most commonly utilized for mammalian expression. The choice of expression system depends on the specific research needs and downstream applications.

For voltage-dependent conformational studies, expression in Xenopus oocytes is often preferred as it allows for both voltage-clamp and fluorescence measurements .

What are the recommended storage and handling conditions for recombinant C15orf27/TMEM266?

Proper storage and handling of recombinant C15orf27/TMEM266 are critical for maintaining protein activity:

Storage recommendations:

• Store at -80°C for long-term stability

• Avoid repeated freeze-thaw cycles by aliquoting the protein

• Typical stability is 12 months from the date of receipt under proper storage conditions

Handling recommendations:

• For applications in cell culture, filtering before use is recommended

• Typical buffer conditions include 25 mM Tris-HCl, pH 7.3, 100 mM glycine, 10% glycerol

• Formulations may vary depending on supplier and specific research needs

How can voltage-clamp fluorimetry be used to study conformational dynamics of C15orf27/TMEM266?

Voltage-clamp fluorimetry has been instrumental in characterizing the conformational dynamics of C15orf27/TMEM266 in response to voltage changes. This technique involves:

• Expression of cysteine-substituted mutants in Xenopus oocytes

• Labeling with fluorescent probes (e.g., TAMRA-MTS)

• Simultaneous measurement of voltage and fluorescence changes

In a significant study, researchers created cysteine substitutions between the external ends of S3 and S4 helices and labeled them with TAMRA-MTS. Eight positions showed accessibility to extracellular solution with measurable fluorescence changes upon membrane depolarization .

Key findings:

• Fluorescence increases (dequenching) were observed following membrane depolarization

• ΔF/F changes ranged from 1% to 5% with nearly linear F-V relations

• Some positions (e.g., P194C, W198C) showed both rapid and slow phases of fluorescence changes

• Using cut-open configuration for optimal clamp speed, increases in fluorescence occurred with a time constant of 130 μs

What methodological approaches can be used to study voltage sensitivity of C15orf27/TMEM266?

Several approaches have been employed to study the voltage sensitivity of C15orf27/TMEM266:

• Chimeric protein construction: The S4 helix from C15orf27/TMEM266 can be transplanted into Hv1 or Shaker Kv channels to test its voltage-sensing capabilities. This approach demonstrated that the S4 helix is capable of sensing membrane voltage within the context of other S1-S4 domains .

• Site-directed mutagenesis: Creating specific mutations, particularly in the S1-S4 domain, can reveal the contribution of individual residues to voltage sensing. This approach has helped identify key amino acids involved in voltage-dependent conformational changes .

• Zinc modulation studies: Extracellular Zn²⁺ has been shown to regulate conformational dynamics of C15orf27/TMEM266, providing a useful tool for investigating voltage-dependent structural changes .

• Electrophysiological measurements: While C15orf27/TMEM266 does not form functional ion channels, electrophysiological techniques combined with fluorescence measurements help characterize voltage-dependent conformational changes .

How does C15orf27/TMEM266 respond to voltage changes despite not forming a functional ion channel?

C15orf27/TMEM266 contains a functional voltage-sensing domain (S1-S4) similar to those found in voltage-gated ion channels, but it does not conduct ions. Studies have revealed that:

• The protein undergoes distinct conformational rearrangements in response to changes in membrane voltage

• Two types of conformational changes occur:

  • Rapid responses following membrane depolarization

  • Slower secondary conformational changes in some mutants (e.g., P194C, W198C)

• The protein shows nearly linear F-V relationships over a ±200 mV range of membrane voltages, which is unusual compared to conventional voltage-sensing domains

These properties suggest C15orf27/TMEM266 may function as a voltage sensor for cellular processes rather than as an ion channel itself .

What is the role of zinc in regulating C15orf27/TMEM266 function?

C15orf27/TMEM266 contains a zinc-binding site that plays a critical role in regulating its conformational transitions:

• Extracellular Zn²⁺ ions have been shown to regulate the conformational dynamics of the protein

• The zinc-binding site specifically regulates the slow conformational transition

• This regulation may be physiologically significant, as zinc is an important neuromodulator in the brain, particularly in regions where C15orf27/TMEM266 is expressed

The zinc modulation provides a potential mechanism for fine-tuning the voltage-sensing properties of C15orf27/TMEM266 in response to physiological conditions .

What is known about C15orf27/TMEM266 orthologs across species?

C15orf27/TMEM266 shows conservation across mammalian species, suggesting important functional roles:

The high sequence conservation, particularly in the voltage-sensing domain, suggests functional importance across mammalian evolution .

Has C15orf27/TMEM266 undergone positive selection during mammalian evolution?

While the search results don't provide specific information about positive selection for C15orf27/TMEM266, evolutionary analysis of mammalian genomes has identified genes that underwent positive selection during evolution of humans and model organisms (mouse, rat, chimpanzee, and dog) .

Researchers interested in evolutionary aspects of C15orf27/TMEM266 might consider:

• Analyzing dN/dS ratios across mammalian sequences

• Applying site models and branch-site models to detect selective pressures

• Focusing on the voltage-sensing domain to determine if it has been subject to purifying or positive selection

This type of analysis could provide insights into the evolutionary history and functional importance of C15orf27/TMEM266 across species.

How can C15orf27/TMEM266 be used in blocking experiments with antibodies?

Recombinant protein control fragments of C15orf27/TMEM266 can be valuable tools for blocking experiments with corresponding antibodies:

Protocol for blocking experiments:

• Use a 100x molar excess of the protein fragment control based on antibody concentration and molecular weight

• Pre-incubate the antibody-protein control fragment mixture for 30 minutes at room temperature

• Proceed with IHC/ICC or WB experiments using the pre-incubated mixture

This approach helps verify antibody specificity by demonstrating that pre-incubation with the target protein blocks antibody reactivity in subsequent experiments .

What genetic association studies have implicated C15orf27/TMEM266 in human diseases?

Genome-wide association studies have identified genetic variants in the 15q24.3 region, where C15orf27/TMEM266 is located, as potentially associated with non-syndromic orofacial clefts (NSOFCs):

• A variant in this region showed significant association with non-syndromic cleft palate only (NSCPO)

• Interestingly, this variant was associated with both NSCPO and non-syndromic cleft lip with palate (NSCLP) but with opposite effects on risk

• Functional annotation of risk alleles within this region, coupled with established roles of candidate genes in periderm development, embryonic patterning, and regulation of cellular processes, supports their involvement in palate development

These findings suggest potential developmental roles for genes in this region, which may include C15orf27/TMEM266, although direct causality would require further functional studies.

What advanced methodological approaches can be used to study the potential roles of C15orf27/TMEM266 in neuronal function?

Given C15orf27/TMEM266's expression in cerebellar neurons and its voltage-sensing properties, several advanced approaches could be employed to investigate its neuronal functions:

• CRISPR/Cas9-mediated gene editing: Creating knockout or knock-in models to study the effects of C15orf27/TMEM266 deletion or specific mutations on neuronal function

• Optogenetic manipulation: Combining voltage indicators with optogenetic tools to correlate C15orf27/TMEM266 conformational changes with specific neuronal activities

• Super-resolution microscopy: Investigating the precise subcellular localization and potential co-localization with other synaptic proteins

• Electrophysiological recordings in neuronal preparations: Examining how modulation of C15orf27/TMEM266 (e.g., through zinc application) affects synaptic transmission and plasticity

• Single-molecule FRET: Studying conformational dynamics of C15orf27/TMEM266 at the single-molecule level to capture heterogeneity in voltage responses

What are the critical controls needed when performing experiments with recombinant C15orf27/TMEM266?

When conducting experiments with recombinant C15orf27/TMEM266, several critical controls should be implemented:

• Expression controls: Verify expression levels and proper localization of recombinant protein using Western blotting and immunofluorescence

• Negative controls for fluorescence studies:

  • Uninjected Xenopus oocytes

  • Oocytes expressing wild-type protein without introduced cysteine residues for labeling

  • These controls ensure that observed fluorescence changes are specific to the labeled protein

• Controls for voltage-dependent effects:

  • Test multiple voltage protocols to distinguish voltage-dependent from time-dependent effects

  • Include experiments at holding potentials where voltage sensors should not be activated

• Specificity controls for antibody experiments:

  • Pre-absorption controls using recombinant protein fragments

  • Isotype controls for immunohistochemistry

• Tag effect controls: Compare tagged and untagged versions to ensure that protein tags (e.g., C-Myc/DDK) do not interfere with function

How should researchers address potential experimental artifacts when studying C15orf27/TMEM266 conformational dynamics?

When studying conformational dynamics of C15orf27/TMEM266, researchers should be aware of potential artifacts and implement strategies to address them:

• Electrochromic effects: The rapid kinetics and nearly linear ΔF/F-V relationships observed in some studies could be attributed to electrochromic origins. Researchers should consider:

  • Testing multiple fluorophores with different properties

  • Controlling for direct effects of electric fields on fluorophores

  • Performing control experiments with charge-neutralizing mutations

• Fluorophore environment effects: The fraction of fluorescence quenched by tryptophan residues can change with protein conformation. Controls should include:

  • Testing multiple positions for fluorophore attachment

  • Mutating nearby tryptophan residues to assess their contribution to observed signals

• Expression level variations: Different expression levels can affect protein behavior. Researchers should:

  • Quantify and normalize for expression levels

  • Test multiple expression systems to ensure consistent results

• Mutation effects on protein structure: Mutations introduced for functional studies might alter protein structure. Consider:

  • Using complementary methods to assess structural integrity

  • Testing multiple mutations at the same or nearby positions

  • Using conservative substitutions when possible