Generation and regulation of the cell resting membrane potential - implications for function

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

All cells have a resting membrane potential (RMP), due to the unequal ionic distribution across the membrane. Cellular integrity and function is closely coupled to the RMP with key processes including, for example, proliferation, secretion, and migration triggered by changes in membrane voltage. Hence, manipulation of the cell RMP has applications for health and disease, for example in cancer where cells have been shown to have RMPs that differ from their normal, healthy counterparts. The cell RMP is most readily recorded by microelectrode methods while specific fluorophores have been developed that measure ion concentrations within cells. Working with researchers equipped with expertise in electrophysiology, cell culture and live-cell fluorescence imaging, the aim of this rotation is to enable the student to sample these cutting-edge methodologies. The RMP in various cell lines will be measured in response to altered ionic gradients across the membrane to compare theoretical and experimental values. The dominant ionic species contributing to the cell RMP will also be determined using well-characterised ion channel blockers. A component of the training will involve the use of live-cell confocal imaging (at QMC) using known fluorophores that track potassium ions. Understanding key concepts of membrane biophysics will enable deeper mechanistic insight into thegeneration of the RMP and manipulation of cellular function.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M008770/1 01/10/2015 31/03/2024
1803030 Studentship BB/M008770/1 01/10/2016 20/01/2021
 
Title Possible signalling pathway mechanisms involving ATP in the pathogenesis of preeclampsia. 
Description Possible mechanisms of ATP as a factor in PE. This model describes the potential mechanisms linking the contribution of eATP to the pathogenesis of PE via the activation of endothelial cells in blood vessels of fetal or maternal origin. Elevated eATP levels stimulate monocytes, Tlymphocytes, neutrophils and endothelial cells. Increased circulating levels of ATP may be caused by other DAMPs being excreted in the maternal circulation for example STBMs and EVs which may travel into the mother's ci 
Type Of Art Image 
Year Produced 2019 
Impact This review article has been cited by the following book Chapter One (page 1-28) named: "Endoplasmic reticulum stress in the cellular release of damage-associated molecular patterns" in the "International review of cell and molecular biology" with a journal impact factor in 2019 of 2.04. DOI: 10.1016/bs.ircmb.2019.11.006. 
URL https://www.ncbi.nlm.nih.gov/pubmed/31424615
 
Description In terms of achievements as a result of the work funded through this award, I have been able to investigate the vasoactive effects of increasing concentrations of ATP and BzATP (P2- purinergic agonists) in isolated human placenta chorionic plate arteries using a well-established laboratory technique named wire myography. This work showed that each cumulative concentration response elicited by ATP starts with vasoconstriction followed by vasorelaxation. Also, each subsequent increase in ATP concentration in all cases gave rise to a smaller amplitude of the contraction which suggests that there could be a desensitization effect taking place. This effect was seen in chorionic plate arteries with both small (260µm) and large (1200µm) internal diameters. Furthermore, BzATP did not elicit any reproducible vasoactive effects on isolated arteries of the chorionic plate.
Important validation experiments have been performed involving the P2X7 receptor quantification using western blotting in three different human tissue types: trophoblast, stem villous arteries and myometrium. These studies show the P2X7 receptor expression in placental tissues has a consistent feature namely the presence of a 50kDa band on all blots. As the expected molecular size of some target proteins in this case the P2X7 receptor which is 75kDa was relatively close to this non-specific one, it was necessary to minimise or remove this 50kDa band. The western blotting data shows that the 50kDa non-specific IgG band has been almost fully eliminated from all homogenised tissues by improving the research methods of an immunoprecipitation protocol using an anti-human IgG (whole-molecule) agarose antibody.
The human placenta is a vital fetal vascular bed that ensures nutrient and oxygen transfer to the fetus. Establishing a reliable chorionic vessel system in vitro will contribute greatly to our understanding of placental vascular function. Furthermore, experiments focused on producing a co-culture model using primary human umbilical vascular endothelial cells (HUVEC) and freshly isolated chorionic plate artery smooth muscle cells (CPA-SMC) were conducted. CPA-SMCs were able to attach to HUVEC without overwhelming the monolayer. Both cell types continued to express their individual cell specific markers including junctional occupancy of VE-Cadherin. Our data suggests that the CPA-SMC /HUVEC model of chorionic vessels may be useful for in vitro complementary studies.
In terms of new research networks and collaborations, I have been able to establish an excellent research partnership between the University of Nottingham and Victor Babes National Institute of Pathology in Bucharest as the Institute was the host organisation for my professional internship for PhD students placement programme. I have also published a review article named: "Inflammation- The role of ATP in pre-eclampsia" in the Microcirculation Journal. In terms of laboratory and computational skills developed during the project, some key techniques that I learned and will be extremely useful for designing and conducting further research were the following: western blotting, wire myography, primary and cancer cells culture, flow cytometry, immunohistochemistry, immunocytochemistry, ELISA, RT-qPCR, florescence microscopy, confocal microscopy, SysMIC, computational systems biology, Image analysis with Fiji.
Exploitation Route The findings of this study might be taken forward by investigating the migratory capacity of smooth muscle cells (SMC) isolated from chorionic plate artery of human placenta under a confluent human umbilical vein endothelial cells (HUVEC) monolayer by using live confocal imaging. Further permeability studies could be performed using HUVEC and SMC cells by creating a reliable chorionic vessel system in vitro. This will contribute greatly to our understanding of placental vascular function. Also, non-hydrolysable ATP analogue compounds may be used to investigate the involvement of purinergic P2 receptors and potentially the P2X7 receptor in the regulation of placental vasculature in the context of wire myography using small diameter chorionic plate arteries, as well as in the circumstance of an in vitro barrier formed by HUVEC and SMC cells. Furthermore, a comprehensive comparison between stem villous arteries and chorionic plate arteries originating from the human placenta may be performed when it comes to investigating the vasoactive effects of non-hydrolysable ATP analogue compounds. The findings of this study might be taken forward by organisations particularly in the field of pharmaceutical and medical biotechnology.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
URL https://www.ncbi.nlm.nih.gov/pubmed/31424615
 
Description Collaboration between the University of Nottingham and Victor Babes National Institute of Pathology 
Organisation Victor Babes National Institute of Pathology Bucharest
Country Romania 
Sector Academic/University 
PI Contribution I have been contributing to this collaboration by sharing my expertise in the area of cell culture, assessing the levels of plasma membrane damage to a squamous cell carcinoma cell line under the influence of various inhibitor compounds used in the treatment of basal-cell carcinoma. I will also share my knowledge with regards to designing and performing laboratory experiments involving an MTS assay which is a calorimetric method for sensitive quantification of viable cells in a cell proliferation assay under the influence of various inhibitors of the hedgehog (Hh) signalling pathway. In terms of intellectual input, I have designed the template of 96-well plate based assay regarding vehicle controls and inhibitor concentrations to be tested on the cell line according to the IC50 concentration values.
Collaborator Contribution Victor Babes National Institute of Pathology have provided the access to the cell culture facilities including the usage of all equipment needed for the culture of the squamous cell carcinoma cell line. The staff have provided me with the cells and culture reagents as well as all inhibitors. I have been granted access to the use of a protein microarray platform for the purposes of high throughput screening of inflammatory markers originating from serum/plasma of patients diagnosed with squamous or bazo-cellular tumours. This partnership has been established as part of my professional internship for PhD Students. Also academic staff in the Immunology Department have been supporting me in the designing of all cell proliferation and protein microarray experiments as well as introducing me to using all of the laboratory equipment involved during the professional internship. The project will bring data regarding inflammatory circulatory markers in non-melanoma as possible biomarkers for monitoring non-melanoma tumours. All work will be presented at the National Immunology Conference and results published in biomedical journal. Furthermore, as part of the project, Victor Babes National Institute of Pathology are contributing financially towards the cost of laboratory consumables as part of the internship with overall £20,000. This includes the cost of protein microarray slides.
Impact This project will bring data regarding inflammatory circulatory markers in non-melanoma as possible biomarkers for monitoring non-melanoma tumours. All results from this work will be presented at the National Immunology Conference and results published in a biomedical journal.
Start Year 2020
 
Description Facing the journalists: communicating your research through the media 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Media (as a channel to the public)
Results and Impact This is a one day Media Workshop was designed specifically for PhDs and early career researchers who need to communicate their work, or comment on related topics, in the non-specialised media. This was a one-day workshop course featured discussion, individual and group exercises and an on camera interview. As part of this course, the trainers, ex-BBC journalists with decades of training experience, prepare exercises and interviews tailored for each participant's area of study, so are able to offer the training to mixed groups from different backgrounds. This interactive workshop gave rise to identifying the elements of a good media story, through the challenges of dealing with the media and the competing pressures of academic and journalistic methods to final on-camera interviews and playback analysis. Throughout, trainees are given examples, templates and tips on how to tackle the exercises, to increase their confidence and broaden their understanding. The objectives of this short course were: 1. Be able to identify newsworthy, 'media-friendly' elements in the research 2. Know how to 'pitch' the research to the media 3. Know and be able to apply techniques for taking control of interviews and for answering difficult questions 4. Have considered how to approach media engagement including giving expert comments and interviews to print media, radio and TV in order to communicate via the media with increased confidence. In terms of outcomes from this activity, I have been able to practice explaining my research to a non-specialist audience and to reflect on the performance in terms of the on-camera interview. The journalists have asked me further questions after the interview expressing their interest in finding out more about my research.
Year(s) Of Engagement Activity 2017
URL https://training.nottingham.ac.uk/Guests/GuestCourse.aspx?CourseRef=GSTCPR
 
Description Faculty Postgraduate Research Forum (Medicine and Health Sciences) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The Faculty Postgraduate Research Forum is an annual showcase of the economic and societal impact of the research being undertaken by doctoral students within the Faculty of Medicine and Health Sciences. Doctoral students from across the Faculty are invited to submit an impact statement of their research project and prepare an impact poster highlighting the economic and societal impact of the research being undertaken. On the day of the Forum, all students will also deliver a '2 minutes of Impact' presentation to the faculty before an afternoon of networking and poster browsing. I have been asked to prepare an e-poster in this case and many of my peer colleagues from across the Faculty have asked me questions about the project. There were 94 doctoral students presenting the socio-economic impact of their research projects. I have won a prize of the best peer nominated e-poster.
Year(s) Of Engagement Activity 2019
URL https://www.facebook.com/UoNGraduateSchool/photos/-mhs-faculty-pgr-forum-june-2019-award-winners-the...