Team:Valencia/Parts/Characterization

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== '''Aequorin characterization''' ==
<br>
<br>
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==General information==
 
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AEQ is a gene which encodes for aequorin, our luminiscent protein.
AEQ is a gene which encodes for aequorin, our luminiscent protein.
 +
It's a photoprotein isolated from luminescent jellyfish (like various ''Aequorea species'' like ''Aequorea victoria'') and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different eukariotes. Nowadays, there are different aequorin types, depending on the target organism.
 +
<br>
[[Image:aequorin.GIF|300px|center]]
[[Image:aequorin.GIF|300px|center]]
-
 
+
<br>
-
It's a photoprotein isolated from luminescent jellyfish (like various Aequorea species like Aequorea victoria) and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different superior eukariotes. Nowadays, there are different aequorin types, depending on the target organism. <br>
+
Cells containing this part are able to sintetize apoaequorin, the apoprotein of 22 kDa. That apoprotein cannot produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca<sup>2+</sup>, full aequorin emits light. The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF-hand motifs that function as binding sites for Ca<sup>2+</sup> ions. When Ca<sup>2+</sup> occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO<sub>2</sub> (as we explain in Wetlab overview). As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.
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The aequorin which we are working with has been introduced in our yeasts by a plasmid called pEVP11/AEQ, wich encondes aequorin sequence showed below. Cells containing this plasmid are able to sintetize apoaequorin, the apoprotein of 22 kDa, and keep it in their citoplasm. So, that apoprotein can't produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca2+, full aequorin emits light.<br>
+
-
The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF-hand motifs that function as binding sites for Ca2+ ions. When Ca2+ occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO2 (as we explain in Wetlab overview). As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.<br>
+
==Sequence==
==Sequence==
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Aequorin sequence is (primer binding sites are underlined in green):<br>
Aequorin sequence is (primer binding sites are underlined in green):<br>
Line 23: Line 26:
==Characterization==
==Characterization==
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===Chemical input.===
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The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell’s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following:
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<br>
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In order to make our yeasts produce light, we firstly reproduce experiments made by Viladevall et al, After a lot of different tries, we finally could characterize the luminiscence curve in a discontinuos luminometer.  
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[[Image:Comparació koh.jpg|center|520px]]
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[[Image:eq1.jpg|center]]
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As we can see in the graphic, a peak of light is emited about 450 seconds before adding 60 microliters of KOH to 170 microliters of medium with WT transformed yeasts. Although we were almost sure that the mechanism that triggered that flash of light was the expected, we found properly make the same experiment with different kind of controls and make sure we were not observing any artiffact:
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 +
Where ''g'' is the conductance associated with the channel, ''V'' is the transmembrane potential and ''E<sub>Ca</sub>'' is the Nernst potential, related to the different ionic concentration inside and outside the cell.
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* '''Mid1''': one mutant for a Calcium channel. Light is not observed because Ca2+ can’t enter into the cell and bind to the aequorin-coelenterazine complex.
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Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance ([[Image:gbarra.jpg]]) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time:
 +
 +
[[Image:eq2.jpg|center]]
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* '''Cch1''': another mutant for Calcium channel, so the absence of light can be explainned in the same way.
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VDCCs are well characterized in excitable cells such as neurons, where the value of [[Image:gbarra.jpg]] is known:
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<center><html><a href="http://partsregistry.org/wiki/images/c/c2/Gneurons.jpg"><img src="http://partsregistry.org/wiki/images/c/c2/Gneurons.jpg"></a></html></center>
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* '''EDTA''': Aulthough every compound necessary for the reaction is present (including Ca2+ channels) light is not emited because EDTA is a divalent ion quelant, so Ca2+ is quenched and not useful for the emission.
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Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its  plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results:
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* '''KCl''': another negative control. The Absence of the -OH group prevents the oppening of calcium channels and makes yeasts produce no light.
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<center><html><a href="http://partsregistry.org/wiki/images/9/90/Gyeast.jpg"><img src="http://partsregistry.org/wiki/images/9/90/Gyeast.jpg"></a></html></center>
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Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation:
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But we wanted to characterize in detail this kind of response.
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===Chemical stimulation===
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To complete the work with the chemical input, we though KOH amounts could influence in the quantity of emited light, so we repited the experiment with different concentrations of KOH.
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<br>
-
 
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Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons –light- are emitted during a short period of time. This graph shows yeasts’ response to the addition of 30 µL of KOH:
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[[Image:Caracterització KOH.jpg|center|thumb|700px| Light emitted under diferent concentrations of the chemical input]]
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<center><html><a href="http://partsregistry.org/wiki/images/1/1b/Koh.jpg"><img src="http://partsregistry.org/wiki/images/1/1b/Koh.jpg" width="600px"> </a></html></center>
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As we can see, the volume of added KOH (from 15 microliters to 120) is related to the luminiscent peak. Although there is not directly proporcional, luminiscence intensity is increased when we increase the quantity of KOH we put in the sample (always 170 microliters of medium with yeasts).
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+
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Characterizasing the response to the KOH we also found interesting to determinate the reproducibility of the process.
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[[Image:Repetibilitat KOH.jpg|700px]]
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Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin.
 +
 +
[[Image:Caracterització KOH.jpg|center|700px]]
 +
It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 30 microliters each (where arrows show):
 +
 +
[[Image:Repetibilitat KOH.jpg|center|600px]]
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By adding 30 microliters of KOH at the time where the rows indicates us, we discovered that before the first peak, cells couldn’t return to the basals levels, and every new shock make yeasts produce light in higher levels than the last one.
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Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph:
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===Electrical input===
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[[Image:Comparació koh.jpg|center|600px]]
<br>
<br>
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When the experiments with an alkali input showed us that yeasts were able to produce light because of their transformation, we tried with our ambitious goal: stimulate calcium channels with an electrical input.
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===Electrical stimulation===
 +
<br>
 +
Calcium entrance to cytosol –and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows):
-
We reproduced the mentionated Arinyo’s protocol, incubating the transformed yeasts with coelenterazine, but changing the KOH by electricity. Surprisingly, we found that  light was also produce in a very similar way. We tryied with different times and voltages in order to find the optim conditions for a big peak of light. Some of our graphics are theese:
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====Discontinous measurements====
 +
The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots.
 +
<center>
 +
{|
 +
|[[Image:1,5V_5st.jpg‎|300px]]||[[Image:1,5V_10s.jpg‎|300px]]
 +
|-
 +
|[[Image:4,5V_5s.jpg‎|300px]]||<html><a href="http://partsregistry.org/wiki/images/6/6d/10_10.jpg"><img src="http://partsregistry.org/wiki/images/6/6d/10_10.jpg" width="320px"></a></html>
 +
|}
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</center>
 +
 +
The time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks.
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[[Image:1,5V 5st.jpg|center|thumb|700px| Light emitted when 1,5V are applicated during 5 seconds]]
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[[Image:6V_variats_disc.jpg‎‎|600px|center]]
 +
 +
That’s probably because a big exposure time of electrical input damages and kills the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium entering in the cell.
 +
In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don’t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock.
-
[[Image:1,5V 10s.jpg|center|thumb|700px| Light emitted when 1,5V are applicated during 10 seconds]]
 
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[[Image:4,5V 5s.jpg|center|thumb|700px| Light emitted when 4,5V are applicated during 5 seconds]]
 
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[[Image:10V 1s disc.jpg|center|thumb|700px| Light emitted when 10V are applicated during 1 second]]
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Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained:
 +
 +
[[Image:Manteniment_resposta_disc.jpg‎|600px|center]]
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[[Image:10V 2s.jpg|center|thumb|700px| Light emitted when 10V are applicated during 2 seconds]]
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Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls (water, SD medium, SD with coelenterazine without yeasts, yeasts without coelenterazine, and addition of EDTA) were used to prove that light came from aequorin-transformed yeasts. Here it is an experiment with 4,5V, 5s long, excitation:
-
[[Image:24V 0,5s.jpg|center|thumb|700px| Light emitted when 24V are applicated during 0,5 seconds]]
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[[Image:Comparació disc.jpg|center|600px]]
-
We realised that the time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks.
+
====Continous measurements====
 +
The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements).
-
That’s probably because a big exposure time of electrical input damages and killes the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium enetering in the cell.
+
[[Image:10V_2s.jpg‎|600px|center]]
 +
[[Image:24V_0,5s.jpg‎|500px|center]]
-
In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don’t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock.
 
-
[[Image:6V variats disc.jpg|500px]]
+
In the following graph each 5 minutes we shocked (arrows) different strains with 16V during 5 seconds. WT aequorin-transformed yeasts and mutant strains (''cch1'' and ''mid1'') are compared. SD medium with coelenterazine and yeasts without coelenterazine are included as negative controls:
-
This graphic clearly show us that using a same voltage, we obtain a better response with the shortest time of the electrical input.  
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[[Image:Valencia_Grafica_continu_2_pics_series.jpg|750px|center]]
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Our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It was not very probable, because the peak observed was produce near 400 seconds before of the stimulus. But, another time, when cells without coelenterazine or mutants are used, we see no light.
+
<br>Again, light production is lower in mutant cells, also in the first peak of light -that we were not able to see with the discontinuous luminometer-, as calcium ions enter through VDCCs. A sharp peak of light is produced when the stimulus is applied, but our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It is not very probable, because the peak observed was produced during near 400 seconds after the stimulus. Another time, when cells without coelenterazine or mutants were used, we saw no light.
-
[[Image:Wt cont.jpg|center|thumb|700px| Our yeasts with coelentrazine ]]
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For the sake of completeness, the different strains and conditions can be seen here, one by one:
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[[Image:Wt-coe cont.jpg|center|thumb|700px| Our yeastse without coelenterazine]]
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Our yeasts with coelentrazine, LEC at a glance!
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[[Image:Wt cont.jpg|center|700px]]
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[[Image:SD+coe cont.jpg|center|thumb|700px| Medium with coelenterazine]]
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[[Image:Cch1 cont.jpg|center|thumb|700px| Mutant yeasts, deficients in calcium channels]]
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[[Image:Comparació disc.jpg|center|thumb|700px| Comparation of the results]]
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Studying the repetibility of the process, this is a little different from the chemical stimulus, but the system has a similar behaviour, and we can stimulate several times the same sample getting a response. However, every next shock produces a fewer peak of light. We hace two hypothesis: one of them is that a part of our yeasts die meanwhile the electrical stimulus. The other one is that coelenterazine is not reusable, so a proportion of it runs down in every emission of light.
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[[Image:Manteniment resposta disc.jpg|center|thumb|700px| Here, we can see that the process can be repeat consecutively]]
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[[Image:Combinació.jpg|center|thumb|700px| Comparation of the repetibility between our yeasts, our yeasts without coelenterazine, medium whit coelenterazine and mutants]]
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===SCREEN===
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<br>
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Using that information and ability of our yeasts, we decided to design a bio-screen, where every single pixel was composed of a group of luminiscent cells and individualy stimulated with a cable. We could, then, control which pixel gets iluminated, forming the image/picture we want (whose resolution depends on the number of pixels we have).
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This simple mechanism is the first example of electronic communication between computers and single celled organisms. Thus, our engineered yeast are a state-of-art bioelectronic device.
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Our yeasts without coelenterazine, no light emission at all
 +
[[Image:Wt-coe cont.jpg|center|700px]]
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'''It is just like a bacterial photographic system, but it's digital.''' Within seconds, instead of hours, you can get an image formed of living cells.
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Medium with coelenterazine: no LEC, no light emitted!
 +
[[Image:SD+coe cont.jpg|center|700px]]
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And, the chose the calcium signaling because it is the fastest known modality of signaling in biology, and will allow for a fast refreshing rate of the screen.
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Mutant yeasts, deficients in calcium channels: very little light emission, due to the inefficient calcium channels
 +
[[Image:Cch1 cont.jpg|center|700px]]

Latest revision as of 01:29, 22 October 2009



Aequorin characterization


AEQ is a gene which encodes for aequorin, our luminiscent protein.

It's a photoprotein isolated from luminescent jellyfish (like various Aequorea species like Aequorea victoria) and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different eukariotes. Nowadays, there are different aequorin types, depending on the target organism.

Aequorin.GIF


Cells containing this part are able to sintetize apoaequorin, the apoprotein of 22 kDa. That apoprotein cannot produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca2+, full aequorin emits light. The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF-hand motifs that function as binding sites for Ca2+ ions. When Ca2+ occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO2 (as we explain in Wetlab overview). As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.

Sequence

Aequorin sequence is (primer binding sites are underlined in green):

Aeqseqval.JPG

Characterization

The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell’s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following:

Eq1.jpg


Where g is the conductance associated with the channel, V is the transmembrane potential and ECa is the Nernst potential, related to the different ionic concentration inside and outside the cell.

Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance (Gbarra.jpg) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time:

Eq2.jpg

VDCCs are well characterized in excitable cells such as neurons, where the value of Gbarra.jpg is known:

Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results:

Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation:

Chemical stimulation


Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons –light- are emitted during a short period of time. This graph shows yeasts’ response to the addition of 30 µL of KOH:

Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin.

Caracterització KOH.jpg

It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 30 microliters each (where arrows show):

Repetibilitat KOH.jpg

Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph:

Comparació koh.jpg


Electrical stimulation


Calcium entrance to cytosol –and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows):

Discontinous measurements

The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots.

1,5V 5st.jpg1,5V 10s.jpg
4,5V 5s.jpg

The time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks.

6V variats disc.jpg

That’s probably because a big exposure time of electrical input damages and kills the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium entering in the cell. In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don’t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock.


Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained:

Manteniment resposta disc.jpg

Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls (water, SD medium, SD with coelenterazine without yeasts, yeasts without coelenterazine, and addition of EDTA) were used to prove that light came from aequorin-transformed yeasts. Here it is an experiment with 4,5V, 5s long, excitation:

Comparació disc.jpg

Continous measurements

The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements).

10V 2s.jpg
24V 0,5s.jpg


In the following graph each 5 minutes we shocked (arrows) different strains with 16V during 5 seconds. WT aequorin-transformed yeasts and mutant strains (cch1 and mid1) are compared. SD medium with coelenterazine and yeasts without coelenterazine are included as negative controls:

Valencia Grafica continu 2 pics series.jpg


Again, light production is lower in mutant cells, also in the first peak of light -that we were not able to see with the discontinuous luminometer-, as calcium ions enter through VDCCs. A sharp peak of light is produced when the stimulus is applied, but our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It is not very probable, because the peak observed was produced during near 400 seconds after the stimulus. Another time, when cells without coelenterazine or mutants were used, we saw no light.

For the sake of completeness, the different strains and conditions can be seen here, one by one:

Our yeasts with coelentrazine, LEC at a glance!

Wt cont.jpg

Our yeasts without coelenterazine, no light emission at all

Wt-coe cont.jpg

Medium with coelenterazine: no LEC, no light emitted!

SD+coe cont.jpg

Mutant yeasts, deficients in calcium channels: very little light emission, due to the inefficient calcium channels

Cch1 cont.jpg