Properties and function of TRPC proteins in vascular smooth muscle using transgenic mice

Lead Research Organisation: St George's, University of London
Department Name: Basic Medical Sciences

Abstract

The aim of our research is to discover molecules that control the diameter of blood vessels. These molecules will be future drug targets for prevention and treatment of cardiovascular disease (CVD).

A recent survey showed that CVD such as high blood pressure, heart attack, and stroke, cause 40% of all deaths in the UK. Also, CVD costs us over £29 billion a year in healthcare, and loss of productivity due to lost work days. With an aging population, these figures are likely to rapidly increase. In light of these facts, we need new drugs to prevent and treat VD.

Not enough flow of blood causes cells to not receive enough nutrients (e.g. oxygen), and to build up more waste products (e.g. carbon dioxide), which leads to cells not working properly. CVD often leads to blood vessels contracting too much, which contributes to a reduction in blood flow. Therefore it is important to find out how blood vessels contract. We study muscle cells found in the walls of blood vessels, which important in making blood vessels contract.

It is known that calcium is an important molecule in making these muscle cells to contract. Our research investigates proteins, called TRPC channels, which are involved in increasing the concentration of calcium inside cells. TRPC channel proteins are pores which are found in the membrane that surrounds cells. Molecules cause these pores to open and close, which allows calcium to move into cells.

TRPC channels are important in controlling how blood vessel contract, and are believed to be involved in contributing to high blood pressure, which is a major risk factor for CVD. The present work will investigate members of the TRPC channel protein family, TRPC1 and TRPC5, in blood vessels from mice, which are an excellent model for understanding what happens in human blood vessels.

Our research we greatly advance knowledge on how blood vessel contract. This will ultimately help in the development of new treatments for CVD. It will also help understand other diseases, which involve TRPC channels and changes in calcium levels inside cells.

Technical Summary

Canonical transient receptor potential (TRPC) proteins are a family of non-selective Ca2+-permeable cation channels (TRPC1-C7) expressed in a plethora of cell types. TRPCs are activated by stimulation of G-protein-coupled and tyrosine kinase receptors, and form Ca2+ influx pathways involved in many cellular functions.

In native vascular smooth muscle cells (VSMCs), TRPCs are likely to be heteromeric structures composed of different TRPC subunits, which contribute to vascular disease by inducing excessive vasoconstriction and migration, growth and proliferation of VSMCs. TRPCs and associated signalling molecules are legitimate drug targets for prevention and treatment of cardiovascular diseases.

Studies on TRPCs have generally used anti-sense/small-interfering RNAs and dominant-negative technologies to 'knock-down' TRPCs in cultured VSMCs, and VSMC-derived cell lines. These studies have provided useful information on 'what can happen', but do not indicate 'what does happen' in native conditions - cultured cells and cell-lines have different cellular environments, and also may not express different TRPCs compared to native VSMCs. We address these issues by investigating single TRPC currents in acutely isolated VSMCs from TRPC transgenic mice. These approaches will reveal properties conferred by individual TRPCs in native vascular environments, and changes in TRPCs following knock-out of TRPC subunits.

Our hypothesis is that TRPC1/TRPC5 subunits confer distinct properties to TRPCs in native VSMCs; small channel conductance, stimulation by agents that deplete internal Ca2+ stores, dependence on protein kinase C (PKC) and phosphoinositols (PIs) for channel gating. We will compare TRPCs in TRPC1-/- and TRPC5-/- VSMCs as these channels are thought to form functional heteromeric TRPC1/C5 channel structures. Moreover, we intend to study the contribution of TRPC1 and TRPC5 proteins to alpha1-adrenoceptor-induced vasoconstrictions.

Planned Impact

In the UK, cardiovascular disease (CVD) accounts for 40% of all deaths. Moreover, CVD costs the country over £29bn, in healthcare costs (£16 bn) and loss of productivity (£13 bn). With inclusion of worldwide data, it is not surprising that cardiovascular research aimed at identifying new therapeutic targets represents multiple beneficiaries from academic, public and commercial environments. Our proposal will investigate such potential therapeutic targets, e.g. TRPC channel proteins and associated signalling molecules.

It should also be remembered that TRPC channels are expressed in many cell types, and are reported to have important physiological and pathological functions in an array of body systems. Therefore, TRPC channel proteins represent therapeutic targets for other diseases, which further increase the number of potential stakeholders supported by our research.

Who will benefit from this research?
- Research groups in the UK/worldwide academic institutions involved in studying the molecular properties and function of TRP channels/Ca2+ signalling pathways in the cardiovascular system, and in other biomedical disciplines.

- Pharmaceutical companies involved in studying TRP channels/Ca2+ signalling pathways as potential therapeutic targets for development of treatments for diseases. For example, Novartis has published on the role of TRPC channels in respiratory diseases, Pfizer on TRPV1 channels in pain and cough reflexes, and GSK on TRPV4 and vascular/endothelin and bladder function.

- Providers of medical treatments, e.g. NHS and medical care insurance companies. CVD costs the UK about £16bn in healthcare.

- The public treasury. CVD costs the UK about £13bn in work productivity, resulting in a significant reduction in tax revenue.

- The health of the population, both within and outside the UK.

How will they benefit from this research?
It is proposed that TRPC1 channel activity is linked to a range of physiological and pathological phenotypes. The majority of studies have been carried out using cultured cells or cell-lines, and little is known about the function of TRPC1 proteins in native tissues. Moreover, few studies have investigated the function of TRPC channels in TRPC1-/- mice, and no studies have investigated the molecular properties and function of TRPC channels in VSMCs from TRPC1-/- mice using single channel recording techniques. Consequently, stakeholders listed above will benefit from our proposal as,

- Our proposal will reveal new understanding and novel perspectives on the functioning of an important ion channel subunit involved in multiple cellular functions, which is studied in worldwide academic institutions and pharmaceutical companies.

- Understanding more about the molecular properties, activation mechanisms and functions of TRPC1 channels will identify new therapeutic targets - not only the channel proteins, but the associated signalling molecules involved in activating/regulating channel activity. This is likely to influence commercial concerns. As with all scientific discoveries, the timescale of these influences translating into therapeutics at the clinic is likely to be 10-15 years.

- TRPC1 has been implicated in a number of diseases, which have a significant impact on the mortality and morbidity of the population. In an aging population, the frequency of many of these chronic diseases, requiring long-term treatment, will not doubt increase. Development of new therapeutic targets and strategies are therefore essential for continued health of the population.

- An aging population with associated increases in chronic conditions are major financial implications for any society. New, more effective, treatments offering increased health benefits and better value for money are of obvious importance to governments.

Publications

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publication icon
Shi J (2016) Store depletion induces Gaq-mediated PLCß1 activity to stimulate TRPC1 channels in vascular smooth muscle cells. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description Calcium is very important for controlling how our cells work. We have found a new way that calcium can be transported into our cells. We believe that this transport pathway is important for controlling how our blood vessels become constricted, which is what happens when we have high blood pressure. In the future we may be able to block this new calcium transport pathway to treat high blood pressure.
Exploitation Route Our data provide the strongest evidence so far that TRPC1 proteins form store-operated channels, and that these channels have a novel activation pathway involving PIP2, PKC, and MARCKS. We have also made the fundamental discovery that store-depletion is coupled to this TRPC1 channel activation process through STIM1 inducing PLC activity - this a novel and potentially high impact finding. We therefore believe that our results make a significant contribution to understanding TRP channel function in vascular smooth muscle and other cell types, which will be important for other areas of investigation by many different research groups.
Sectors Chemicals

Education

Healthcare

Pharmaceuticals and Medical Biotechnology

 
Description It is hoped that our data will impact societies understanding of vascular physiology and pathology. This will lead to better treatments for cardiovascular disease, improved population morbidity and reduce mortality. Furthermore it will have a positive impact of economic prosperity of the population.
First Year Of Impact 2014
Sector Chemicals,Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Investigation into Calcium-sensing receptor mechanisms in the vasculature
Amount £136,595 (GBP)
Funding ID FS/13/10/30021 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 09/2016
 
Description Role of PIP2-binding protein MARCKS on voltage-gated Ca2+ channels and vascular contractility
Amount £114,469 (GBP)
Funding ID FS/15/44/31570 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2015 
End 09/2018
 
Description STIM1-mediated PLC activity and TRPC1 channel activation in vascular smooth muscle
Amount £365,834 (GBP)
Funding ID BB/M018350/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2015 
End 06/2018
 
Title Lentiviral shRNA techniques 
Description Use of lentiviral technology to transfect shRNA sequences into vascular smooth muscle cells. Manipulation of vectors to express different STIM1 structures 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? No  
Impact This has enabled us to carry out molecular interventions for the first time in out laboratory 
 
Title PIP2 sensor 
Description GFP-PLCdelta-PH is a plasmid used to detect PIP2/IP3 levels 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The use of GFP-PLCdelta-PH has enabled us to measure PLC activity in primary cultured vascular smooth muscle cells. This techniques had a major impact in our recent work showing that store-depletion induces PLC activity 
 
Title Primary cell culture 
Description We have developed a cell culture techniques whereby vascular smooth muscle cells are maintained in low (1%) serum conditions for up to 7 days. These cells retain their contractile properties 
Type Of Material Cell line 
Provided To Others? No  
Impact The development of these cell culture conditions has enabled us to carry out molecular interventions (transfection of plasmids, siRNA, biosensors etc.) on vascular smooth muscle cells with a contractile phenotype 
 
Description Role of P2X receptors in vascular smooth muscle 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide technical support for wire myography experiments, helped write manuscripts
Collaborator Contribution Provided research assistant collaboration
Impact 2 papers
Start Year 2013
 
Description Role of PIP2 on TMEM16A ion channels 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of common ideas, development of new techniques - expression of PIP2 sensors, dot-blots analysis to investigate ion channel/PIP2 interactions
Collaborator Contribution Expression of TMEM16A in HEK293 cells
Impact 2 papers, several abstracts
Start Year 2013
 
Description Role on Orai1 proteins in vascular smooth muscle function 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution Collaboration with the Harvard Medical School to determine the role of Orai1 proteins in controlling vascular tone. This international and potentially very important collaboration is a direct result of support from my BBSRC grant.
Collaborator Contribution Supplying Orai1 +/- mice, for production of orai1-/- genotypes
Impact Recent invited speaker at Young Life Scientists Symposium on 'Physiology and Pharmacology of TRP channels', King's College London, 4th October, 2014
Start Year 2013
 
Description Attendance at Research Day, St. Georges, University of London, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Presented poster to academics, postgraduates, undergraduates (mainly MBBS, BSc Biomedical Sciences, MPharm students) from St. George's, University of London

No
Year(s) Of Engagement Activity 2014
URL http://www.researchday.sgul.ac.uk/
 
Description Attendence at 42nd Symposium on hormones and cell regulation (European Society of Endocrinology) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation on ' TRPC1 SOCs are activated by a STIM1-PLC-mediated pathway indpendently of Orai1 in vascular smooth muscle'
Year(s) Of Engagement Activity 2017