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

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.

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.

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.