Potassium-Channels in Guard Cells - From Phenomenon to Molecule. III. Structural Analysis
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| License | No Open Access License: German copyright law applies. This film may be used for your own use but it may not be distributed via the internet or passed on to external parties. This film contains music to which the collecting society GEMA holds the rights. | |
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Transcript: English(auto-generated)
00:25
Erwin Neher and Klaus Röschke have provided the groundwork for studying biological processes from the phenomenon to the molecule.
00:42
In contrast to the spontaneous growth of crystals, the growth, movement and differentiation of living cells is mediated by protein molecules.
01:16
The first plant potassium channels were characterized and cloned from stomatal guard cells.
01:23
Iron channels, proteins of the plasma membrane, mediate the exchange of solutes and information between cells and their environment. The molecular blueprint, and thus the structure of these proteins, is stored in the nucleus. Following identification of the gene structure of the first potassium channels,
01:44
large-scale genome projects continuously deliver DNA sequences encoding new potassium channels. This has become possible with the aid of a new generation of automatic sequencers and ultra-fast computers. It's possible to generate an evolutionary tree of potassium channels which includes bacteria, fungi, plants and even humans.
02:14
The distance between individual branches of the tree indicates the genetic diversity between the various potassium channels and symbolizes the relationships within the three groups of organisms.
02:24
When comparing the amino acid sequences of all the potassium channel proteins, an alternation between variable and conserved regions is visible, the latter indicated by vertical bars.
02:43
One characteristic is shared by all potassium channels, irrespective of their origin. From a mixture of cations of almost similar size, they are able to selectively transport the potassium ion. The smallest common element is represented by the amino acid sequence glycine-tyrosine-glycine,
03:01
a motif found in all potassium channels, including those of bacteria and even viruses. Within this motif and the adjacent region, amino acids will be replaced to test their function in the guard cell potassium channel. The replacement of a single amino acid residue, for example a charged by
03:37
an uncharged or a hydrophobic by a hydrophilic, is called site-directed mutagenesis.
03:43
This is done at the level of the DNA. A plasmid carrying the potassium channel cDNA serves as a template in a polymerase chain reaction. The single molecular steps within a mutagenesis cycle are now demonstrated.
04:07
In the first step, the plasmid DNA is denatured. Hydrogen bonds break. And the double-stranded DNA separates, or melts.
04:21
After the reaction temperature has been lowered, an oligonucleotide, which carries the desired mutation, attaches to the single-stranded DNA. The free three-prime end of the oligonucleotide serves as a primer for a DNA polymerase, which copies the circular DNA leading strand.
04:46
This increase in DNA chain length is called elongation. A plasmid hybrid consisting of mutated and non-mutated DNA evolves. The first mutagenesis cycle is completed.
05:12
The second cycle is initiated by melting of this hybrid.
05:25
During the second annealing process, another primer attaches to the mutated single strand. It fits perfectly. During the elongation process, the DNA polymerase again elongates the free three-prime end of the primer.
05:47
The product of the second mutagenesis cycle is a mutated double-stranded potassium channel cDNA. This process is repeated many times to amplify the selectively mutated potassium channel cDNA.
06:06
It is transcribed in vitro into a copy cRNA and is ready to be used for a functional analysis. To test the function of transport proteins, an expression system is used which itself possesses no or only a very few ion channels.
06:35
In this respect, oocytes of the South African clawed toad Xenopus levis have become well established.
06:47
After the viability of the oocytes has been checked, they are prepared for injection with cRNA, encoding the mutated potassium channel gene. With the help of micromanipulators and an automatic injection system, a small capillary impales the oocyte and injects the cRNA.
07:13
In this way, a population of oocytes that express the mutated potassium channel gene is generated.
07:21
In a second oocyte population, the unmodified potassium channel gene of the wild type is injected. In the following analysis, a comparison between both populations is possible. Inside the cell, the potassium channel mRNA is recognized by ribosomes and translated into protein subunits.
07:48
They assemble and fuse with the plasma membrane of the oocyte. As early as one to two days after injection of the potassium channel
08:01
cRNA, the electrical properties of the oocyte membrane are dominated by potassium channels. Oocytes are placed in the cavity of a perfusion cuvette and exposed to a potassium solution. Using the two electrode voltage clamp technique, potassium currents flowing
08:21
through many hundred thousand potassium channels expressed simultaneously are monitored. For this purpose, a voltage and current electrode is carefully inserted into the oocyte. This is accomplished with micromanipulators. The voltage electrode on the left measures the membrane potential.
08:43
The current electrode on the right injects exactly as much current as is needed to maintain a given test potential. The potassium currents resulting from the changes in membrane potential are recorded by an amplifier and displayed.
09:01
A computer controls the measurement and analysis of the data. Now we follow the potassium current into the oocyte using the wild type channels as an example. In response to stepwise changes in membrane potential to negative values, with the channel selected here, a time-dependent potassium current is elicited.
09:32
On replacement of potassium in the test solution by sodium, no currents are observed, even with strong polarization of the membrane to minus 160 millivolts.
09:44
This indicates that the potassium channel is impermeable to sodium ions. On repeating this experiment with different channel mutants, one finds that among them some mediate ionic currents even in a sodium solution.
10:07
The selectivity filter is, as it were, leaky and allows sodium to move through the channel in addition to potassium. This experiment shows that quite a restricted region of the protein forms a selectivity filter of the ion-conducting pore.
10:28
Now we will determine the three-dimensional structure of the channel protein by crystallization. Using microorganisms, which can be grown to a dense suspension in fermenters, the protein in question is enriched millionfold.
10:44
Here a membrane protein with a purple bacterium, Rhodobacter spheroidis. In the first step, the bacteria have been sedimented in a flow-through centrifuge to separate them from the surrounding growth medium.
11:06
On the plastic membrane during centrifugation, a bacterial sediment several millimeters thick has formed. In the sediment, the density of the bacteria and thus of the membrane protein to be isolated is increased manyfold.
11:30
With a spatula, the cell concentrate is transferred to a beaker and weighed.
11:43
For the subsequent isolation and crystallization of the membrane protein, one needs several grams of sediment. The following steps take place in a cold room to prevent the extremely labile membrane proteins from disintegrating.
12:09
By chromatographic methods, they will now be separated from one another. After solubilizing the cell membrane in a detergent-containing buffer, the solution
12:24
contains the protein in question as well as many other membrane proteins. In the last step, the highly enriched membrane protein is separated from other proteins by ion exchange chromatography. It appears as a dark band and while moving through the column, shown here in time-lapse, separates from the other proteins, which are discarded.
12:51
The channel protein is eluted from the column and directed to a fraction collector.
13:03
Now the basic requirements for the subsequent crystallization of the protein are fulfilled. The protein concentrate is pipetted into special crystallization dishes.
13:39
Afterwards, at constant temperature, water is gradually removed from the protein suspension.
13:47
In this way, protein molecules come into close contact with one another and aggregate into protein crystals. A time-lapse sequence helps to illustrate this process, which takes several days.
14:02
The crystal consists of many millions of identical channel proteins. When the protein in the suspension is used up, the crystal growth stops. Crystals that are now one to three millimeters in diameter are prepared for the analysis of the crystal structure.
14:26
A selected crystal is drawn into a measuring capillary. The ends of the capillary are sealed with wax to protect the very fragile protein crystal from disintegration caused by air humidity.
14:48
The three-dimensional structure of the protein crystal is now determined by X-ray analysis. For this purpose, the glass capillary containing the crystal is aligned with the X-ray source.
15:09
The detector is positioned right next to it. Subsequently, the X-ray beam hits the crystal. In response, the protein crystal diffracts the X-ray beam.
15:24
A detector screen monitors the diffraction pattern. The crystal is now rotated stepwise around its axis. The X-rays, which are normally not visible, are illustrated here. For each angle, the diffraction patterns are computed in relation to each other.
15:44
Like the orthopedist, who identifies the exact position of bones and vertebrae from an X-ray image, the crystallographer calculates the position of every individual amino acid within the protein crystal from the diffraction pattern. In contrast to the doctor, however, the crystallographer is not satisfied with millimeter differences.
16:05
He has to reach a resolution of one to two angstrom, which is a ten-millionth of a millimeter, to solve the three-dimensional structure of a potassium channel. Here is the result of such an analysis on the potassium channel from the bacterium Streptomyces lividans.
16:25
As visible from the top view, the channel is made up of four identical subunits. In the center of the channel complex is situated the ion conducting pore, in which the potassium ions are located. After rotation through 90 degrees, it can be seen that these four subunits are made up of alpha-helical stretches
16:46
of about 17 to 21 amino acids, and anchor the membrane protein within the membrane. Following removal of the front and rear subunits, the potassium-selective region of the pore is visible.
17:02
The constriction in the channel pore is formed by the three amino acids glycine, tyrosine, glycine. The motif that all potassium channels have in common. This essential selectivity filter for potassium ions is stabilized by two pore helices.
17:26
The path of a potassium ion through the channel pore is exemplified on a simple model. The negative membrane potential draws a potassium ion into the pore, where it binds to the selectivity filter
17:43
until a second potassium ion binds, which, due to electrostatic repulsion, causes the first ion to be released. Negative charges at the entrance to the pore prevent the entry of anions, here shown in silver.
18:00
The selectivity for potassium in relation to other cations is the result of specific binding sites at the channel filter. Due to repulsion amongst the potassium ions, fluxes of the order of 10 to the power of 7 ions per second are obtained. When mutations occur, ion channel function is impaired. Sometimes, the channels even lose their activity.
18:27
In plants, these genetic defects can result in malfunction of guard cells, growth defects in the shoot and root. And in humans, they're related to hereditary diseases, like arrhythmia, epilepsy, and cystic fibrosis.
18:50
Thus, the structural analysis of ion channels is an essential prerequisite in, for example, the development of new approaches to therapy and the optimization of plant breeding.
19:03
The information for realization of these projects can be gained from protein crystals. The structural analysis, together with biophysical and molecular genetic analyses, will facilitate the step from molecule to application.