Mechanisms and physiological significance of the transfer of biologically relevant gases across cell membranes

• Applicant: Privatdozent Dr. Volker Endeward
• Subject Area: Anatomy and Physiology
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 253516712

Aquaporins (AQP) and Rhesus proteins have been shown to cause increased membrane CO2 permeability. This agrees with molecular dynamics simulations showing that these membrane proteins constitute channels for CO2. Such channels appear biologically relevant in view of our recent observations that membranes with a cholesterol content of ~ 50 mol%, as is normal for most cell membranes, exhibit an unexpectedly low CO2 permeability, about two orders of magnitude lower than membranes without cholesterol. Part 1 of the present project aims to elucidate the molecular mechanism of CO2 transfer through AQP, especially the contribution of the AQP water pore vs. the central pore of the AQP tetramer. Our model will be the isoform AQP5, whose central pore is occluded in the presence of the phospholipid PS, but is open if PS is not present. AQP5-mediated CO2 permeabilities in the presence and absence of PS will allow us to quantitate the contributions of the water pore of the monomer and of the central pore of the tetramer. Part 2 of the project is based on preceding observations showing that cultured cell lines that possess a low oxidative metabolism express no gas channels and exhibit low membrane CO2 permeability. We will now study cells from tissues with high oxidative metabolism and/or high rates of CO2-O2 exchange. We will measure the membrane CO2 permeability of mouse red cells, rat cardiomyocytes and mitochondria. It is expected that these cells have high CO2 permeabilities, and these permeabilities will be related to the membranes´ expression of gas channel proteins. The underlying general hypothesis is that cells with normal membrane cholesterol possess a quite low CO2 permeability, and that cells functionally requiring high CO2 permeabilities achieve this by expressing protein gas channels in their membranes. In part 3 of the project the presently unknown membrane permeabilities of three other gases of great biological interest, O2, NO and CO, will be studied. The questions to be answered are a) what are the permeabilities of membranes devoid of gas channels? Does cholesterol cause a similarly drastic reduction of gas permeability as it does in the case of CO2? and b) are the intrinsic gas permeabilities of cell membranes low enough to render membrane gas channels physiologically meaningful? We will produce hemoglobin-loaded artificial lipid vesicles with cholesterol in the absence and presence of reconstituted gas channels, as well as human red cells, to study this by stopped-flow spectrophotometry. Part 4 of the project is dedicated to the systemic consequences of gas channel deficiency on the O2-CO2 transport system of mice. Using the Helox technique we will measure maximal O2 consumption of normal and mice deficient in AQP, Rhesus protein and both proteins. A reduction of maximal O2 consumption could be due to a reduced permeability for O2 or CO2 of the membranes of red cells, cardiac muscle cells, lung, skeletal muscle or mitochondria.

• DFG Programme: Research Grants