* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Biol2174 Ionic composition of cells
Cell growth wikipedia , lookup
Cellular differentiation wikipedia , lookup
Magnesium transporter wikipedia , lookup
Cell culture wikipedia , lookup
Extracellular matrix wikipedia , lookup
Cell encapsulation wikipedia , lookup
Mechanosensitive channels wikipedia , lookup
Cytokinesis wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Signal transduction wikipedia , lookup
Membrane potential wikipedia , lookup
Cell membrane wikipedia , lookup
Biol2174 Cell Physiology in Health & Disease Lectures 5 & 6: Ion Composition & Methodology School of Biochemistry & Molecular Biology Ionic composition of cells • All cells, from those in your brain, to those of the simplest unicellular organisms living in the sludge at the bottom of the sea, maintain an intracellular (i.e. inside) composition different from that of the extracellular (i.e. outside) medium (plasma or sea water in the two examples given). • The reason that they are able to do this is that they are enclosed within a membrane, that limits and controls the movement of molecules and ions between the intracellular and extracellular solutions. Table 11-1 Molecular Biology of the Cell (© Garland Science 2008) Ionic composition of cells A couple of points to note • Although the concentration of Ca2+ in the cytosol is very low, several organelles serve as intracellular stores of Ca2+ and have a high concentration of this ion. The flow of Ca2+ into the cytosol, from either the extracellular solution or the intracellular stores serves as a ‘signal’. • The fact that in most cells K+ diffuses out of the cell (down its concentration gradient) faster than Na+ diffuses into the cell has the effect of producing an inwardly negative electrical potential (i.e. a ‘membrane potential’). In some cells, under some circumstances, the rate of diffusion of Na+ increases, causing the membrane potential to become more positive. Measuring fluxes Commonly by using a radiolabeled form of the solute of interest. e.g. 3H, 14C, 32P or 33P labeled compounds 22Na+ for Na+ 42K+, 43K+, 86Rb+ for K+ 36Cl- for Cl45Ca2+ for Ca2+ * * Influx: * * * * Efflux: * * * e.g. Uptake of [33P]phosphate into isolated malaria parasites 33P i Parasite uptake (distrib. ratio) Na+ Pi 125 mM Na+ 0 mM Na+ Time (min) Saliba & Martin, Bröer, Henry, McCarthy, Downie, Allen, Mullin, McFadden, Bröer & Kirk Measuring ion concentrations Measuring ion concentrations Fluorescent indicators Measuring ion concentrations Fluorescent indicators variable Measuring ion concentrations Fluorescent indicators - Dye leakage Measuring ion concentrations Using indicators ratiometrically [Ca2+]× Isosbestic Excitation wavelength Indicators that can be used in this way are referred to as ‘ratiometric’. 340 nm Resting [Ca2+]i Peak [Ca2+]i Recovery 380 nm % initial fluorescence intensity 140 120 100 80 60 40 340 nm 354 nm 380 nm 20 0 0 5 10 15 Time (seconds) 20 25 30 Measuring ion concentrations Fluorescent indicators dye loading Measuring ion concentrations HELA (cancer) cells stained with the Calcium indicator Fura-2. Two images of fluorescence at different excitation wavelengths (340nm & 380nm, intensity color coded) were recorded and subsequently the ratio was calculated to achieve a Calcium concentration distribution independent of indicator concentration. Finally the color coded Calcium distribution image was overlaid on a standard microscope image of the cells. www.till-photonics.de Electrophysiological methods Measuring the membrane potential Electrophysiological methods The patch-clamp technique • Developed by Erwin Neher and Bert Sakman in the late 1970s and early 1980s. Revolutionised cell physiology. Neher and Sakman were awarded the Nobel prize for Medicine in 1991. • In their initial experiments they pressed a firepolished glass micropipette up against the membrane of an intact cell and found that this enabled them to measure the electrical currents flowing through individual channels in the patch of cell membrane encompassed by the pipette as the channels open and closed. • The first single-channel current records were published in 1976. • However the sensitivity and hence the scope of this original method was limited by the large background leak of current (i.e. ions) between the pipette and the membrane. • The real breakthrough was reported in 1981 when Neher and Sakman showed that with the application of a gentle suction (applied by mouth!) clean glass pipettes fused to clean cell membranes to form a seal of unexpectedly high resistance and mechanical stability. • They called the seal a gigaseal since it can have an electrical resistance as high as tens of gigaohms (giga = 109). Electrophysiological methods The patch-clamp technique Molecular methods Isolating membrane proteins Figure 10-30 Molecular Biology of the Cell (© Garland Science 2008) Reconstituting isolated transport proteins Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008) The Xenopus oocyte expression system transport transport or uptake of radiolabel. transport * * * * Radiolabel uptake Expression cloning using Xenopus oocytes The right panel shows the different cloning steps graphically. The left panel represents the transport assay with the expected results at the corresponding stage of the project. The strategy in brief: 1) Isolation of total poly (A)+ RNA from rabbit kidney followed by injection and transport assay (I) in oocytes. 2) Fractionation of the mRNA, injection of the fractions and transport assay (II). 3) Construction of a cDNA library from the active rabbit kidney mRNA fraction. Division of the library into different pools of clones. Synthesis of in vitro transcribed mRNA, injection and transport assay (III). 4) Subdivision of the pool(s) as described above down to one single clone (IV). Hydropathy plots ¾ Used to find clusters of hydrophobic amino acids ¾ May indicate that the polypeptide in question is a membrane spanning protein ¾ For an 㱍helix, a 20 amino acid sequence will just span the membrane ¾ So an hydropathy plot is used to search for 20 amino acid stretches of hydrophobic amino acid residues Hydropathy plots Figure 10-21 Figure 10-22a Molecular Biology of the Cell (© Garland Science 2008) Hydropathy plots Figure 10-22b Molecular Biology of the Cell (© Garland Science 2008) Crystallisation of membrane proteins Structure determination Structure determination • Roderick MacKinnon and colleagues published the first structure of an ion channel in 1998. • The MacKinnon laboratory chose to work with bacterial potassium channels which could be churned out in large quantities by E. coli and also turned out to be extremely stable. • Even on being taken out of the membrane environment, they remained tetrameric and crystallized well in a detergent mixture. • Since this initial triumph the structure of an increasing number of other ion channels, and transporters has been solved. • More on this in following lectures. Structure determination MacKinnon was awarded the Nobel Prize in Chemistry (at the age of 47) in 2003.