Discussion of the Current Theories on the Specificity of Pruriceptive Neurons and the Chemical Mediators of Pruritus

Pruritus (itch) is a sharp tingling sensation resulting from a potentially harmful stimulus, chronic and neurogenic itch are persistent bouts of itching with (chronic) and without stimulus (neurogenic). Although scratching is a reflex response to itching, if the irritation is extensive there is more likely to be a pain reflex before scratching which potentially means that scratch is to alleviate irritation rather than to protect against what has caused it. Due to the overlap with pain, historically it was thought that itch was the same mechanism and pathway as nociception but interpreted differently as it is a lower intensity (Frey, 1922), in effect a warning that pain may occur if the body stays exposed to the stimulus but is not immediately dangerous. This has largely been rejected in favour of the suggestion that there are specific neurons controlling itch (Schmelz et al., 1997). However, the overlap between itch and pain mean a specific itch system may be too simple and the neurons may conduct nociceptive signals as well (selective theory). The following review will tackle the evidence for the selectivity theory with regards to peripheral neuron types and discuss the potential mediators and corresponding receptor subtypes responsible for itch signalling at the peripheral terminals of dorsal root neurons.

Identification of itch signalling neurons

It has been hypothesised that pruritus is transmitted along primary afferents specific to itch (Schmelz et al., 1997). Yosipovitch contested specificity theory with claims that noxious heat and scratching inhibit itch as it changes ones perception from itch to pain. This claim was supported by their results identifying the intensity of itch decreasing by 0.9cm (P<0.05) (Yosipovitch et al., 2005). However, the scale used was a visual analogue scale which although they showed to be reliable in repetition, is not the most accurate way to measure itch intensity because it is designed to measure subjective characteristics. Yet itch can be quantifiably measured in the frequency and duration of scratch methods which have high construct validity, as many species including humans scratch to alleviate itch. Their claim is supported more conclusively in 2015` with the co-expression of itch neuronal markers and TRPV1 (table1) suggesting noxious heat would have an effect on itch signalling (Usoskin et al., 2015). Usoskin’s team identified neurons responding to itch by using RNA-sequencing to group dorsal root neurons into sub-populations based on RNA expression. They identified 13 sub-groups of sensory neurons, with non-peptidergic unmyelinated C-fibres having 3 sub-groups identified (NP1-3)(Usoskin et al., 2015). These groups contained populations of neurons expressing itch receptors. Different populations expressed different itch receptors at varying expression levels (figure1A-D) suggesting that the different populations are involved in different types of itch. Their results identify selectively high expression of somatostatin in NP3 neurons (0.83)(Table1), this was therefore used as a marker for NP3 neurons.

When co-precipitating somatostatin tags with the tag for isolectin-B4, a known nociceptive marker, Usoskin’s team found that IB4 did not co-localise with somatostatin (figure1E/F). The absence of IB4 in NP3 subpopulations has been documented since this discovery (Stantcheva et al., 2016). This seems to suggest that NP3 is not nociceptive and therefore itch specific. However, TRPV1 is also a nociceptive marker and is significantly expressed in NP3 neurons (0.58)(Table1). Therefore, these neurons could be prompted to elicit nociceptive signalling due to TRPV1 presence.

RNA-sequencing can produce false positives due to artefacts (Ozsolak and Milos, 2011). This can lead to mean data that is not representative of the expression of that RNA. Usoskin’s team made efforts to minimise this limitation by setting a threshold for expression for each gene and only considering the genes that exceeded that expression when grouping populations ((Usoskin et al., 2015) supplementary methods).

Itch receptors and itch mediators

Histamine

Histamine was the first identified itch mediator which might be expected due to the alleviation of acute itching by antihistamines, importantly histamine helped explain the antagonistic relationship between itch and pain (scratch alleviates itch). Nilsson’s team used cutaneous field stimulation (nociceptive electrical impulses) to abolish histamine induced itch in an area of skin, 4 hours after, itch intensity was still 32% lower than control (Nilsson and Schouenborg, 1999). Although histamine is used experimentally to induce itch, pathological itch is unaffected by histamine (Klein and Clark, 1999) and expression maps identify only low levels of the histamine receptor Hrh1 in NP2 and NP3 (table1). The low receptor expression and lack of effect in pathological pruritus may explain why acute itch is only a low level irritation. High receptor occupancy would be required to evoke action potentials which may also explain why high local histamine concentrations are required for itch such as in inflammation.

Interleukin-31

Previous studies identified that interleukin-31 (IL-31) was implicated in chronic disease (Dillon et al., 2004; Takaoka et al., 2006). Since then research identified IL-31 as a key mediator of atopic-dermatitis in mice (Grimstad et al., 2009). It took until 2013 to demonstrate that IL-31 induced scratching with a single acute dose (Arai et al., 2013) see in figure2A. Arai observed that lengthy scratching to IL-31 was higher if applied to lesioned skin, supporting IL-31s role in chronic itch from previous studies (figure2B) but no receptor had been conclusively identified. Usoskin et al then uncovered that NP3 neurons expressed the IL31ra receptor (figure1G) in equal abundance to TRPV1 (0.58)(table1). They investigated this further by treating mice with IL-31 and observing the frequency of scratching increase from 10 to 50 (figure2D). Usoskin also states that NP3 neurons are involved in chronic itch, therefore so is IL-31, which supports the work done by Dillon and Takaoka’s teams (Dillon et al., 2004; Takaoka et al., 2006). However, Dillon et al had to overexpress IL-31 and used transgenic mice to introduce IL-31ra into the epithelial cells. These are not usual causes of atopic-dermatitis so construct validity of the model is limited. In addition, although the model showed good face validity as the mice had similar symptoms to the human disease, their results could not be translated into wider context because no empiric evidence was provided for increased scratching, despite it being stated in the text. Takaoka later produced a similar study but including results showing increased frequency of scratch (figure2C), implicating IL-31 in physiological as well as pathological itch.

Serotonin

Serotonin was found to be involved in itch when Weisshaar applied it to human skin (Weisshaar et al., 2004). Serotonin was also shown to increase scratching in vivo (figure1H) and 5HT1f was highly expressed (0.83) in NP3 neurons and scratching increased from 10 to 80 in response to 5HT (figure2D) which supports the claim that serotonin receptors have a functional role in itch (Usoskin et al., 2015).

Morita’s team showed that 5HT7 is coupled to TRPA1 cation channels by expressing 5HT7 or TRPA1 alone in human embryonic kidney cells and showing no inward calcium current, then co-expressing them and observing an inward current (Morita et al., 2015) also see figure3A. This suggests they are coupled but does not confirm that they cause itch or that other ion channels are not involved. So the team produced knockout mice, to show that there was a significant reduction in scratching if either 5HT7 (65s reduced to 25s) or TRPA1 (35s reduced to 5s) was ablated, suggesting both are required for itch (figureB/C). In addition, the TRPA1 knockout suggests that without TRPA1, 5HT7 does not lead to itch therefore is not coupled to other ion channels involved in itch.

The role of another TRP channel, TRPV4, has been implicated in 5-HT mediated itch (Akiyama et al., 2016), yet in vivo, Morita showed reduced scratching time from 35 to 5 seconds in response to 5-HT without TRPA1 suggesting only TRPA1 is sufficient for itch. However, Akiyama’s results show significantly reduced scratching in TRPV4 knockouts (100 bouts reduced to 20) but no difference between wildtype and TRPA1 knockouts (figure3D). This discrepancy (compare figure 3C and D) is not explained in the literature so a follow-up study should be undertaken to identify 5-HT response in vivo and in voltage-clamp experiments of isolated neurons with TRPV4, TRPA1 or both ablated to identify the contribution of each.

Histamine and IL-31 induce itch along with inflammation and serotonin induces itch at micromolar concentration but pain at millimolar concentration (Morita et al., 2015). This could be evidence that itch neurons also respond to pain, however the response could be from separate classes of neurons with the same receptors.

Conclusion

In summary, it is clear that a sub-population of non-peptidergic neurons (NP3) transmit pruriceptive signals. It is still not completely clear whether these neurons are specific to itch signalling or selective for itch. The lack of co-localisation between itch markers and IB4 suggest the neurons are itch specific but involvement of TRPA1, TRPV1 and TRPV4 in pruritus and nociception suggest that the neurons could be prompted to respond to nociceptive stimuli. To conclude, the evidence from these mediators and receptors strongly suggests that itch neurons can conduct nociceptive signals but it is still possible that the neurons are itch specific. A future approach may be to use optogenetics to induce action potentials specifically in itch neurons by introducing photoactivated ion channels under the somatostatin promoter. Then record the in vivo response to identify whether when scratching is induced these neurons become silent or nociceptive.

Hi everyone

Firstly, I know it has been 2 months since I uploaded anything for you all. My dissertation has taken over everything as well as difficult final year exams so I have not had much time to put anything together as it takes a long time to research the posts and then write them up.

I finish in 3 weeks so the plan for after that is to give a bit of an idea about what my dissertation was like and some tips for how to do well if you have one to do soon.

I will also return to writing overviews of areas of neuroscience.

In the meantime, I have a very interesting post on pruritus (itch) signalling which I will be posting later today. So look out for that, it is worth the read.

Highlighting the importance of pharmacogenetics. Follow up from TPMT deficiency

Drug development is plagued by ineffectiveness or toxicity in significant proportions of the population. This is because blockbuster drug dose is based on the results from cumulative response curves. So the dose is decided by (for example) above 80% of the population respond. However, at this point there will be people that are under-responsive and over-responsive at this dose (figure 1). So they will get no effect or get toxicity at this dose. With certain drugs this is fatal, with 100,000 deaths a year from drug toxicity and 4 million serious adverse reaction events. Variations exist through polymorphisms and drug interactions mostly, the following will discuss some contributions to variability in drug response with some experimental examples.

Absorption, distribution and excretion
There is less variation seen in these processes than metabolism but there are some important variations that can play a big role in drug effectiveness. Absorption is generally similar in most people but disease state can cause variability, for example, 32% of patients requiring irinotecan have diarrhoea, therefore oral administration would create considerable variation with those with diarrhoea absorbing a lower dose than those without. This has to be a consideration in drug development because it suggests the drug should not be developed to be delivered via this route.

Drug distribution and excretion also become variable with disease state, distribution rate is increased if as the disease progresses hypertension and tachycardia increase. In contrast, kidney damage and failure in late stages of disease reduce the body’s ability to excrete the drug, allowing it to be active longer thus increase response.

Metabolism

Metabolism activates and inactivates drugs, so changes to metabolism will cause variation in response because reducing either of these will either lose the effectiveness of the drug or increase the action well over the usual response and be toxic. Many drugs are heavily metabolised by CYP450 enzymes and when an enzyme is metabolising one it cannot metabolise the other. This can cause variability in drug response because one person may be on the drug alone whereas anoth may be taking other drugs. This is exemplified by mibefradil which is safe alone but when administered with propranolol, inhibited CYP450 enzymes, preventing the inactivation of propranolol. A study conducted looked at patients taking both and found systolic blood pressure decreasing from over 140mmHg systolic to 60-70mmHg a fatal level. This was found to be because propranolol was not being inactivated by CYP450s and thus acting excessively on the heart. This drug was soon discontinued.

Other than drug interactions inhibiting or increasing cYP450 metabolism of another drug, CYP enzymes also get polymorphisms, relatively commonly. CYP2C9*2 is the mutant allele in 20% of Caucasians, this is a significant number of people with a loss of function of an important enzyme in drug metabolism. Testing of how much variation the presence of this allele causes showed as much as 30% differences in dose of warfarin required for efficacy. This is a large variation because if a full dose is 30% more effective it is also likely to be toxic at this level. This becomes a problem in drug development because it makes it impossible to give an accurate recommended dose and makes it likely the drug will fail in clinical trials due to toxicity. Evidence that these polymorphisms contribute heavily to variation in drug response between humans, comes from studies adding CYP2C9*2 allele to transgenic mice but using a pharmacogenetics approach of genotyping and phenotyping activity and adjusting the warfarin dose given based on the result. They found that doing this required fewer adjustments to the dose and fewer incidences of toxicity due to too high doses. This suggests that CYP polymorphisms cause lots of variations to metabolism, but pharmacogenetics can be used in drug development to get the correct dose much faster and safer and almost eliminate the variation that is seen.

The future for genetic CYP testing could be in producing transgenic mice with CYP mutations as part of animal models to identify what changes in dose are required for each polymorphism. Evidence it is accurate from measuring CYP2D6 polymorphisms to quinidine and measuring the changes in response to doses and changing dose until variability is within acceptable levels.

CYP polymorphisms are not the only causes of variation in response, other important enzymes also cause variation in response between people including thiopurine methyltransferase (TPMT). TPMT*3A-C polymorphisms cause a total loss of function of this enzyme which allows a build-up of active thiopurines this in turn leads to an increase in efficacy and toxicity which eventually leads to myelosuppression. In this case variation has been fatal, because the toxicity is quickly fatal and a full dose to some heterozygote mutants is 50% higher than the dose should be and 15% of people are at least heterozygous for a TPMT mutation. Again the relationship with drug development is that drug cannot be developed to suit most but not all because it can be fatal. This could lead to a loss of millions at clinical trials or even more if it has to be discontinued. This is more evidence that variation in patients has to be accounted for in drug development, therefore pharmacogenetics may be the approach in rug development to create more individualised medicines from drugs that are technically block busters, it should increase success in clinical trials along with reducing variation.

Non-metabolic genetics

Aside from metabolism genetics comes into other variations between patients. The same cancer can have different responses to the same drug in different people. This is also a type of variation in response. Also taken into account is the stage of cancer because as the cancer progresses mutations are random so the same cancer can look very different by stage 4. A good example of variation to the drug is the drug Herceptin for breast cancer. It acts on the HCN2 receptor and is very effective but only in 25% of cases because men do not have the receptor and the receptor can be down regulated early in the cancer. This shows the variation in drug response because the drug is very effective at treating breast cancer, but the receptor is not present in all people or all breast cancers. This causes a problem in drug development because it is obvious the drug is very effective but getting significant results is difficult because of how variable cancers are.


In summary, variation in drug response is seen more commonly in metabolism but absorption, distribution and excretion also lead to variation between people, mainly due to what state the disease is in. metabolism variation exists mainly through one drug changing the enzymes action on another drug or mutations to important enzymes. However, it is important to note that sometimes the genetics of the disorder are variable as in cancer.

Interesting slightly different post

I hope you enjoy the TPMT deficiency post, it is a typical pharmacogenetic interaction and I will post a follow on writing that will take this into a wider context and into the difficulties pharmaceutical companies have when developing new drugs. 

Please give me some feedback on the two posts as if the consensus is good I will do a few more similar things that are not strictly Neuroscience. 

Enjoy! 

Thiopurine S-methyltransferase (TPMT) Deficiency

Introduction:

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the methylation of aromatic or heterocyclic thiol (sulphydryl) compounds (Peng et al., 2008).This function is ubiquitous, therefore the enzyme could be active in methylating toxic compounds as part of immunity such as against bacterial toxins. Its ubiquitous function is mimicked by its tissue expression and expression levels (Figure 1A). RNA-sequencing reveals that not only is it present in all tissues but it is present in specialised areas within those tissues (Figure 1B) and there is little variability between the expression levels in all tissues (Figure 1A and B vertical black lines show range), with the average expression around 0.55 FPKM (Figure 1A and B vertical red lines). The narrow range and ubiquitous nature of this enzyme show that TPMT has been highly conserved throughout all cell types and plays an important role in cell protection. The TPMT protein is coded for by the TPMT gene and has numerous polymorphisms which cause varying levels of loss of function of the expressed protein (Otterness et al., 1997). Interestingly, even those that are homozygous seem to live normal lives regardless of complete TPMT deficiency, unless they are treated during their life with the immunosuppressive thiopurine drugs, which in the absence of TPMT lead to extensive cell apoptosis. This report will discuss the TPMT gene and its mutations and assess the pharmacogenetic interaction between thiopurine drugs and the mutated alleles of this gene. 

TPMT Genotype:

TPMT is located at position 22.3 on the P-arm of chromosome-6 (figure 2a), it is therefore autosomal so a person can be homozygous for one allele or heterozygous. In heterozygotes the two alleles will be expressed equally so the alleles are co-dominant but the majority of the global population are homozygotes (Weinshilboum and Sladek, 1980). Complete TPMT deficiency is autosomal recessive as it requires two copies of the mutant to produce low to absent function, however heterozygotes with one mutant allele will have a varying response to thiopurines. In the general population 89% are homozygous, 11% heterozygous and 0.3% homozygous mutant (Weinshilboum and Sladek, 1980). In 2011, Booth included evidence of 30 alleles (Booth et al., 2011) with relatively few mutations in total suggesting the allele differences are due to the combination of mutations rather than separate mutations. This observation is supported by genetic information from the Uniprot database which also shows a limited number of mutations (Uniprot, 2015). Despite a limited sample, Stanulla provides evidence that TPMT2 and TPMT3A-D account for 95% of TPMT deficiency (Stanulla et al., 2005), which is supported by equivalent studies (Collie-Duguid et al., 1999; McLeod et al., 1999). TPMT*3A is most common in Caucasians with Tai identifying 75% of the sample with TPMT*3A allele (Tai et al., 1996).In contrast, TPMT*3C is the most common allele in Asia (Collie-Duguid et al., 1999; Hiratsuka et al., 2000; Lu et al., 2006) and Africa (McLeod et al., 1999; Ameyaw et al., 1999). TPMT*3C is characterised by a single missense point mutation of tyrosine to cysteine at position 240 due to an adenine to guanine substitution in exon 10 (Figure 2B). TPMT*3A has this same mutation, in addition to an alanine to threonine mutation at position 154 (Figure 2B).



Protein structure and function in wildtype and mutant alleles:

TPMT is a 245 amino acid protein consisting of 10 exons that form 9 beta sheets and 8 alpha helices (figure 3A) that fold to form the tertiary structure in Figure 3B. This figure also shows the binding site for adenosyl methionine and an adjacent binding site for 6-mercaptopurine from Peng’s study, identifying that adenosyl methionine forms at least seven hydrogen bonds here but 6-mercaptopurine only forms one (Peng et al., 2008). This study of the structures and binding sites concurs with the predicted function that adenosyl methionine binds as a methyl donor to the thiopurine, producing methylated thiopurine and adenosyl homocysteine in the reaction (figure 3C) (Peng et al., 2008). The same mechanism is expected for endogenous substances, however, no endogenous substances have been identified. It is possible that TPMT is a cellular defence to exogenous substances that attack through DNA integration. Evidence for this is highlighted by the toxic effects of thiopurine drugs in those with TPMT deficiency. In wildtype, TPMT inactivates prodrug thiopurines before they can be converted to active compounds (figure 3D) (Mlakar et al., 2016). Active compounds such as methylthioinosinemonophosphate inhibit the purine biosynthesis pathway which prevents DNA synthesis and repair or other active metabolites that insert into DNA forming interstrand crosslinks and single strand breaks that lead to apoptosis due to extensive changes in gene expression and DNA damage (figure 3D).

The tyrosine to cysteine mutation in TPMT*3C changes the structure because tyrosine has a large side chain that forms hydrogen bonds and Van der Waals interactions with residues on beta7, beta9 and alpha8. Mutation to cysteine means alpha8 interactions are lost, causing it to pull away (Rutherford and Daggett, 2008). This makes the thiopurine binding site more flexible so the thiopurine is exposed to solution. Making the thiopurine less likely to accept methylation and instead become unbound. In the TPMT*3A allele this structural deformation is coupled with a loss of hydrogen bonding between alanine and tyrosine, the double mutation forms a flattened protein and destabilises the protein causing it to be degraded by proteasomes (Rutherford and Daggett, 2008). Therefore has a half-life of 20 minutes compared to 11 hours in TPMT*3C allele (Rutherford and Daggett, 2008). This seems to be the major contributing factor as to why TPMT activity is almost absent in TPMT*3A homozygotes rather than changes in enzyme action which is the explanation for TPMT*3C allele loss of function.



Clinical features, treatments and diagnosis of TPMT deficiency:

Clinical features of TPMT deficiency are the same as the toxicities of the drugs, only the incidence and severity is higher in those with the disorder so fatality is higher (Dewit et al., 2010). To control the toxicity in those with the deficiency the dose is reduced by 50% for heterozygous and 90% for homozygous mutants (Coenen et al., 2015).

The main adverse effect is myelosuppression, a reduction in erythrocytes (anaemia) and leukocytes (leukopenia) due to the destruction of newly differentiated stem cells in bone marrow (Colombel et al., 2000). The crossover between efficacy and toxicity is evident here because when thiopurines are used to treat lymphoblastic leukaemia (increased level leukocyte derived cells), they act to reduce the number of these cells but the white blood cell loss can be excessive, causing leukopenia. This means there is a narrow therapeutic window. In those with the deficiency, leukopenia is more severe due to the excess active metabolites.

Approaches to diagnosis include phenotype testing which surveys TPMT enzyme activity by adding thiopurines and measuring the metabolite level in a purified sample (Burnett et al., 2014). Or genotype tests which sequence the gene to identify mutations that could change TPMT activity. However, a novel mutation may not be picked up (Burnett et al., 2014).

Treatments tend to deal with the toxicities as there has been no co-agonist identified that can increase the endogenous activity of the non-mutant allele in heterozygotes. Future therapy may be to reduce the degradation rate of TPMT allowing it to have a longer action. However, the most effective way would be through inhibition of the protease pathway which is ubiquitous and heavily relied on in the body. This would likely lead to cell apoptosis due to a build-up of toxic substances, so is not a viable option until another way to reduce its degradation can be found. Myelosuppression can be managed through managing anaemia, by stimulating erythropoietin with Darbepoetin alpha and neutropenia can be rescued with granulocyte colony stimulating factors as well as prophylactic treatments of infection. There has been a suggestion that treating with thioguanine may provide a less toxic alternative to other thiopurines. This has shown to be the case for inflammatory bowel syndrome, unfortunately, the licencing has been rejected due to hepatotoxicity but a derivative with some structural changes could be a future therapy. 

Conclusion:

In summary, the limited treatments and genetic variation of the TPMT gene in the population highlight the need for individualised treatments especially in pharmacogenetics. The wildtype allele functions as a methylating enzyme expressed ubiquitously throughout the body and may have a role in foreign immunity as no endogenous substrates have been identified. Mutant alleles for TPMT lead to damaging effects due to an inability to reduce the cytotoxic metabolites of thiopurine prodrugs and therefore cause fatal infections through myelosuppression. TPMT*3A and C are the most common mutant alleles but TPMT*3A disrupts function primarily due to an increased degradation rate, whereas TPMT*3C has a lack of function due to a change in protein structure. The negative impacts of this drug to mutant interaction are severe but their benefit outweighs the negatives because the majority of the population do not have any mutation and they are used in the treatment of conditions that are fatal.

Sorry about the absence

I know it has been a few months that I have been absent, however it was due to exam revision for January exams and the actually process of taking those exams.

I have a few things lined up for the people that read this blog including an interesting and slightly different post on inheritance and how mutations in metabolic enzymes can be a big problem in the drug development industry. It will begin with a post based on inheritance and then a subsequent post will delve further into its effects in pharmacology.

I am also going to release a few things specifically on neuroscience, which I know most of you like or you wouldn't be reading this.

And for those of you who are students or aspiring students, I am going to keep a short account/diary thing of my dissertation that I will publish on here. As I am doing a laboratory project not a traditional critical review so I will try to talk about lab life and how it combines with writing a dissertation, which will hopefully help some people decide what they want to do when you get here.      (it will most likely apply to other biomedical sciences too so do not think it is just neuroscience labs)

(Also any of you that can, please press the follow button on my blog, I know quite a few of you people out there read it but no-one has followed it so it looks like I am talking to myself).

Thanks

Experimental design: getting from cloning a new gene to finding out its role

You have cloned a new gene X. Patch clamp recording from HEK293 cells overexpressing X revealed that X-transfected cells express large ionic currents when these cells are mechanically stimulated (i.e. by touching the cell with glass rod); no such currents were seen in untransfected cells. Immunostaining of rat tissues with antibodies against X revealed that X is highly expressed in a subset of large dorsal root ganglia and trigeminal ganglia neurons and also in the auditory hear cells. Design the experimental strategy to test if gene X is an ion channel, what other tissues it is expressed in and its physiological role. 

Cells that express ionic currents when one recombinant gene is implanted but no current in native cells means that this gene is likely an ion channel. The current direction gives an indication of the role of the gene as a large downward inflection would suggest a role in depolarisation. However, without further experimental evidence, a whole cell patch clamp recording cannot confirm that gene X is an ion channel. The following will discuss the techniques for identifying whether it is an ion channel, tissue expression and the function of the X protein.

Is it an ion channel and what does it conduct?

Before adding the recombinant channel, a green fluorescence protein tag sequence can be added to the gene. This would allow viewing of the plasma membrane through Total Internal Reflection Fluorescence Microscopy (TIRF) which emits light at an angle where the wavelengths are all reflected away from the specimen but some photons diffuse through and excite fluorophores only on the PM. So there will only be a strong fluorescent signal from the protein if it is present on the PM. If this was not the case it is unlikely to be an ion channel. Using native HEK cells as a control will make for easy comparison.

Ion channels have a distinct single-channel recording profile so a cell-attached patch clamp to isolate one channel could be done and record from the single channel whilst mechanically stimulating the cell. An ion channel would provide a current trace like in figure 1. 

Also this technique discounts the chance that the gene is an ion-pump because a single pump recording is too low to be detected. siRNA should be used to reduce the expression of the mRNA of the gene and then redo whole-cell voltage clamp recordings and compare the first recombinant cell recordings as a control. If the current is significantly reduced it is likely that the gene codes an ion channel. A control using scrambled siRNA can be used which as it has no effect on X-mRNA so if the current still decreases then it is not due to X-mRNA knockdown as this non-specific action is a limitation of siRNA.

Identifying the ions it conducts can begin with adding NMDG, if the current is ablated then the channel is a cation channel. If not, it is likely a chloride channel. A cation channel could be non-selective so the channel can then be treated with tetrodotoxin (Na+), tetraethylammonium (K+) or ruthenium red (Ca2+) separately, if the current is reduced by any of these then the channel conducts that ion. If all do then it is non-selective. However, not all subtypes of each cation channel are inhibited by these blockers, such as NaV1.7. Therefore, the results can be confirmed by removing each cation from solution and measuring the current.

Expression in other tissues:

Immunohistochemistry and electrophoresis can be used in conjunction to support the results of each test. Being as this is a newly cloned gene it is unlikely there is an antibody for it. One could be produced in normal animal-anti-animal technique or an epitope could be genetically introduced into the gene of transgenic species. Tissue slices are taken and stained with the specific primary antibody to bind and then a secondary, fluorescent antibody. The tissue will then fluoresce under microscope if the tissue expresses the protein. Occluding primary antibody acts as a control to show the non-specific binding of secondary antibodies.

SDS-PAGE electrophoresis denatures and adds uniform charge to the proteins of a sample from each tissue. The proteins are separated by size then radio or fluorescent antibodies are used on the film which will appear as a blot under x-ray or microscope if the protein is expressed in the tissue. The control for this will be to normalise the expression level compared to actin as it is expressed at similar levels in all tissues.

Testing the physiological role of gene-X:

It is wise to use siRNA knockdown first because the siRNA can be delivered to the dorsal root ganglion via viral mediated injection and reduce the expression levels of the gene so behavioural and physiological changes can be tested. It is especially useful to do first because the same individual can be used as the control and experimental group which reduces the variability in response and reduces the number of animals being used at this stage. If this stage produces positive results then knockout mice can be used to compare.

Flanking the gene with LoxP and having KO mice only express Cre under the NF200 promoter so the gene would only be lost in large diameter DRG neurons. After doing so it is possible to view any behavioural changes and because it is likely a mechanically activated ion channel, use tests such as Von Frey filament to measure changes to innocuous touch. Post-testing, DRG neurons can be dissected and stained to identify any changes to the neuron physiology.

Evaluation of this experimental design:

The methods being used are ones common to this type of research and yield reliable, reproducible results that are easily interpreted without the need for extensive normalisation calculations. In addition, the experimental design that is laid out uses at least two methods for collecting data so each will support the results from the other or present discrepancies that can be dealt with rather than drawing false conclusions. Also, with fairly few experiments, the tissue expression, which ion is conducted, the mechanism by which the channel is activated (mechanical) and the function can be identified. However, the specific way mechanical stimuli open the channel is not identified.

Transfection of genes is only 10% so large cultures are required for success, also GM and transgenics are expensive and have stringent guidelines for use. TIRF is also an expensive technique. Another issue is that low levels of expression in tissues are not detected by these methods. If required RNA-sequencing could be used instead to detect low levels. Cysteine linked optogenetics would be useful for identifying the channel function but can only be used if the channel is ligand activated, as this channel is mechanical, it cannot be used.

In summary, gene-X can be identified as an ion channel through distinct traces when measuring single channel currents and using siRNA to knockdown the gene to view the change in current. The ion it conducts can be identified by removing specific ions from the solution and measuring the change in current. Electrophoresis will show the tissues expressing gene-X and knocking out the gene will identify the function of the gene through the changes that occur when it is not expressed.