STIM1-mediated PLC activity and TRPC1 channel activation in vascular smooth muscle

Lead Research Organisation: St George's University of London
Department Name: Molecular & Clinical Sci Research Inst

Abstract

A recent British Heart Foundation survey showed that cardiovascular diseases (CVD) such as angina, heart attack, heart failure, and stroke cause one quarter of all deaths in the UK. CVD also costs the UK over £29 billion a year in healthcare plus loss of work days. These figures caused by CVD numbers will increase as the number of elderly people in our population continues to grow. It is clear we need new drugs to treat CVD.

A decrease in the diameter of the open space or 'lumen' of our blood vessels produces high blood pressure and reduces blood flowing to our organs - both these problems increase the risk of us developing CVD. It is known that when muscle cells found in the walls of blood vessels contract too much they decrease the lumen diameter. Our research focuses on understanding what causes these muscle cells to contract too much. By knowing more about these muscle cells and how they control lumen diameter of blood vessels we will help develop new drugs to treat CVD.

The amount of calcium inside these muscle cells is highly associated with contraction of these muscle cells and therefore the lumen diameter of blood vessels. When calcium levels increase too much it causes the muscle cells to excessively contract and reduce the lumen of blood vessels. We investigate proteins, called TRPC1, which form specialised holes called channels that are found in the membrane that surrounds muscle cells. When these TRPC1 channels are opened they allow calcium to be transported into muscle cells. If we could find a way to reduce TRPC1 channels from opening, we could reduce the amount of calcium entering muscle cells, which would reduce contraction of these cells, and prevent the diameter of blood vessels from becoming too small: this would be an excellent strategy for treating CVD.

In this proposal, we plan to investigate what causes TRPC1 channels to open and let calcium into muscle cells using the blood vessels of mice, which are an excellent animal species for allowig us to understand what happens in human blood vessels.

Our research will greatly advance our knowledge on TRPC1 channels and on how the lumen diameter of blood vessels may be controlled. This will directly help in the development of new treatments for CVD. TRPC1 channels also control the level of calcium in cells from other parts of the body such as the brain, kidneys and lungs. Therefore our studies will also help understand diseases involving a variety of other body systems.

Technical Summary

Canonical transient receptor potential 1 (TRPC1) Ca2+-permeable cation channels regulate cytosolic Ca2+ levels involved in diverse functions including muscle contraction, secretion, and gene expression. In vascular smooth muscle cells (VSMCs), TRPC1 channels contribute to vascular disease (VD) by inducing excessive contraction, migration, growth and proliferation. TRPC1 channels are legitimate drug targets for VD.

A strength of this application is that we will study activation mechanisms of endogenous TRPC1 channels in freshly isolated and primary cultured VSMCs, i.e. TRPC1 channels in their native contractile environment. Understanding activation of native TRPC1 channels is important for discovering their role in physiological and pathological processes - essential information in developing TRPC1 channels as pharmacological targets for treatment of VD.

We have revealed that TRPC1 channel activation requires protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PIP2) in native VSMCs; PKC-dependent phosphorylation of TRPC1 increases binding of PIP2, which acts as a gating ligand to induce channel opening. Endogenous vasoconstrictors and agents that deplete internal Ca2+ stores activate TRPC1 channels. TRPC1 channels are therefore defined as store-operated channels (SOCs) - a controversial definition, as it is unclear how store-depletion couples to TRPC1 channel opening.

We will investigate a novel role for stromal interaction molecule 1 (STIM1), a Ca2+ sensor present within internal Ca2+ stores, in activation of TRPC1 channels. Store-depletion induces STIM1 activation, which then stimulates the classical Gq protein/phospholipase C (PLC) signal transduction pathway to drive PKC-dependent TRPC1 phosphorylation and channel gating by PIP2.

There is huge interest in TRPC1 channels, STIM1, and Gq protein/PLC signalling, and therefore we anticipate that our findings will have significant impact on research investigating many organs systems.

Planned Impact

In the UK, cardiovascular disease (CVD) accounts for a quarter of all deaths. Moreover, CVD costs the country over £29bn per year, in healthcare costs (£16bn) and loss of productivity (£13bn). 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. TRPC1 channel proteins and associated signalling molecules such as STIM1-mediated pathways.

It should also be remembered that TRPC1 channels and STIM1 are expressed in many cell types, and are reported to have important physiological and pathological functions in an array of body systems. Therefore, TRPC1 channel proteins and STIM1-mediated pathways 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 TRPC channels/Ca2+ signalling and STIM1-mediated pathways in the cardiovascular system and 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 TRPC3, TRPC6, and TRPV4 channels in cardiovascular and bladder function.

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

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

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

How they benefit from this research?
It is proposed that TRPC1 channel activity and STIM1-mediated pathways are linked to a range of physiological and pathological phenotypes. The majority of studies investigating TRPC1 and associated molecules such as STIM1 have been carried out using expression systems and cell lines, and therefore little is known about the properties of TRPC1 proteins in native tissues. Consequently, stakeholders listed above will benefit from our proposal for the following reasons.

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

- Advancing our understanding of the molecular properties, activation mechanisms and functions of TRPC1 channels will identify new therapeutic targets - not only the channel proteins, but also associated signalling molecules (e.g. STIM1, MARCKS) 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 and STIM1 have been implicated in a number of diseases, which have a significant impact on the mortality and morbidity of the population, e.g. CVD. In an aging population, the frequency of many of these chronic diseases will no doubt increase, which will substantially increase the requirement for long-term treatment and care. 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.
 
Description Investigations into calcium-sensing receptor mechanisms in the vasculature
Amount £130,820 (GBP)
Funding ID FS/17/40/32942 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Project Grant - 'Investigation of MARCKS-PIP2-voltage-gated Ca2+ channels pathway in vascular smooth muscle'
Amount £191,659 (GBP)
Funding ID PG/18/69/33870 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2019 
End 01/2022
 
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 10/2015 
End 09/2018
 
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 Morpholino knockodwn approach 
Description Use of directed morpholino approach to reduce MARCKS expression in vascular smooth muscle cells 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact Morpholino-directed knockdown is a common strategy now used in my laboratory 
 
Title Mutagenesis studies 
Description Use of mutagenesis to investigate store-operated PKC-phosphorylation sites of TRPC1 channels in vascular smooth muscle. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact Mutagenesis studies to investigate store-operated PKC-dependent phosphorylation sites within TRPC1 channels forms an important objective of our new BBSRC submission (Responsive Mode Jan 2019). 
 
Description Effect of TRPC1/4/5 channel blockers on vascular smooth muscle 
Organisation University of Leeds
Department Astbury Centre for Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Through a MTA, Dr Robin Bon has provided novel TRPC1/4/5 channel blockers, e.g. Pico145, for testing in vascular smooth muscle using electrophysiology and wire myography experiments.
Collaborator Contribution Dr Robin provides chemicals, we provide functional experiments using channels in vascular preparations.
Impact Pico145 inhibits store-operated currents in vascular smooth muscle, and also reduces vascular contractility to vasoconstrictors.
Start Year 2018
 
Description Effect of novel TRP channel modulators in vascular smooth muscle 
Organisation Monash University
Department Malaysia Campus
Country Malaysia 
Sector Academic/University 
PI Contribution We are currently testing the effect of novel TRP channel blockers on whole-cell cation currents using the patchc clamp technique in freshly isolated single rat mesenteric artery vascular smooth muscle cells - involvement of Dr Albert and Dr Miguel Martin-Aragon Baudel
Collaborator Contribution The Malaysia campas of Nottingham University, UK have synthesised and provided us with novel TRP channel blockers
Impact Data currently being collected - and grants being written (Newton Fund, The Royal Society)
Start Year 2017
 
Description Attendance at Research Day, St. Georges, University of London, 2015 
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
Year(s) Of Engagement Activity 2015
 
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
 
Description Attendence at International Symposium of Resistance Arteries, Manchester, 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Conference oral presentation at International Symposium on Resistance Arteries, 3rd-6th Sept, 2017, Manchester, UK - Regulation of voltage-gated Ca2+ channels and vascular contractility by MARCKS
Year(s) Of Engagement Activity 2017