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Transcript
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.