Applied pharmacology to biotecnology

CALIBRATION

If we want to link the ratio to the calcium concentration, we need to know (to calibrate

the system):

- the Kd of our sensor

- the minimal ratio (the ratio that we have at 0 calcium concentration)

- the maximum ratio (the ratio at saturating calcium concentration)

- A2: which is the fluorescence intensity at wavelength 2 at saturating

- B2: which is the fluorescence intensity at wavelength 2 when calcium

concentration is zero

This is an example in which we have loaded neurons with fura-2. The calcium

concentration is monitored as changes in colors. We add glutamate to our population: in

neurons, the administration of glutamate induces calcium concentration increase in

soma and dendrites; while if we induce depolarization with KCl we induce a calcium

increase not only in the soma and in the dendrites, but also in the axons. Why if I apply

glutamate I see a difference in the extent of calcium increase within the different

portion of this polarized cell? I have to use multiple labelling, I have to know which is

the nature of my neurite: I use fura-2 as a sensor and I use specific antibodies to identify

dendrites (post synaptic components) and synapsis (pre synaptic components, so the

axons). With this double labelling upon glutamate stimulation we see an increasing

calcium in the soma and in the dendrites (MAP2, microtubular associated protein 2, is a

marker of dendrites). We can conclude that the parts where I have an increase in calcium

concentration upon glutamate stimulation are dendrites. While we can see that axons

(the marker of axons is syp, synaptophysin) experience increase in calcium concentration

only upon depolarization.

NON RATIOMETRIC DYES: FLUO and RHOD

Increasing Ca2+ concentrations and increasing binding to the probe are exclusively

indicated by the increased fluorescence emission of the dye.

By using one single excitation wavelength and emission recording these dyes can be

used with quite simplified microscope set-up. Moreover by using a single wavelength for

excitation other fluorophores can be used to detect/monitor other cellular parameters.

Most non ratiometric dyes are excited with visible wavelengths.

Increasing calcium concentrations and increasing binding to the probe are exclusively

indicated by the increased fluorescence emission of the dye.

Advantages of using indicators that are excited with visible light:

- Confocal microscopes can be used

- Reduced cells and tissues autofluorescence

- Reduced cellular damage and scattering

We have:

- Fluo-3, Rhod-2 and derivatives

- Low affinity indicators: Fluo-5N, Rhod-5N, X-Rhod-5N and derivatives

- Calcium Green, Calcium Orange e Calcium Crimson

- Oregon Green 488 BAPTA

- Fura Red

- Calcein

We don’t use a ratio in this case so we experience all the issue mentioned before, so we’ll

have unequal loading of the dye, focal plane shift, different concentration of the dye, and

so on. However, we can combine two non-ratiometric dyes, ending up with the same

configuration of a ratiometric dye.

An example: fluo-3 and fura red, both excited at 488 nm; the emission of fluo-3 is at 525

nm and it increases at increased calcium concentration, the emission of fura red is at 650

nm and it decreases at decreased calcium concentration. So with the combination of

these 2 dye we obtain a third dye which is a ratiometric one, but we use visible light to

excite these two dyes. These probes have been used to evaluate calcium concentration in

different systems, also in living organisms.

GENETICALLY ENCODED INDICATORS: BIOLUMINESCENCE

AND FLUORESCENCE

We can manipulate cells or organisms in order to induce the expression of genetically

encoded indicators. We have two different tools, bioluminescence and fluorescence

probes.

BIOLUMINESCENCE

Bioluminescence is the emission of photons from a number of photoproteins synthesized

by a number of different organisms (both marine and terrestrial). In bioluminescence

photons are emitted without previous absorption of incoming excitation photons.

Generally the number of photons emitted is quite low, thus bioluminescence signals are

less brilliant with respect to fluorescence signals.

Aequorin

Aequorin is a calcium probe because it is able to bind intracellular free calcium, which is

associated to the emission of a limited number of photons. Aequorin is a photoprotein

isolated from the jellyfish Aequorea victoria. In 1960 the phenomenon of

bioluminescence in the jellyfish Aequorea Victoria was investigated (Shimomura, 1962).

Identification of both aequorin and green fluorescent protein.

Before genetic engineering, for many years the protein was purified from the jellyfish

(avoiding any interaction with calcium ions) and injected into the cells under exam. After

cloning of the cDNA for aequorin its use as a tool to measure calcium in cells strongly

increased also because now the expression of the probe could be specifically directed to

subcellular compartments of interest.

The polypeptide has three calcium-binding domains (EF hand motifs) and, upon calcium

binding, coelenterazine (coenzyme) is released by the molecule with the emission of

2+

photons (very few photons). So, a Ca -induced conformational change of the apoprotein

leads to peroxidation of the coenzyme (the reduced form of the coenzyme coelenterazine

is shown in black and the oxidized form, coelenteramide, is shown in dark blue), which

results in the release of blue light.

The difference between unstimulated and stimulated is high and there is virtually no

background, so it is a super nice sensor for calcium.

Since the generation of the first cDNA encoding for aequorin many constructs have

been generated in order to induce the expression of the protein and make it localize in

different specific cellular compartments (we have the one directed to mitochondria, to

the nucleus/cytoplasm, to the plasma membrane, to the ER and to the sarcoplasmic

reticulum). We can also have some mutations in the sequences of the protein leading to

differences in the emission spectrum, so we can have chromatic variants of the protein.

An example: we have a cell transfected with 2 different constructs of aequorin, one

directed to the lumen of ER and one (a mutated one, a chromatic variant) directed to

cytoplasm; cells are stimulated with histamine, that binds its GPCR and activates the

downstream signalling. Histamine induces the opening of the calcium channels, so

calcium will decrease in the lumen of ER and it will increase in the cytoplasm. So if we

monitor the concentration of calcium in the lumen if ER with our sensor we’ll see a high

fluorescent signal in resting conditions and then, with histamine, we have a reduction of

the fluorescence, meaning a reduction of the calcium concentration. If we monitor

calcium concentration in the cytoplasm, we have zero fluorescence in resting conditions,

but when we apply histamine we have a transient increase of the fluorescence of the

cytosolic probe.

FLUORESCENCE

GFP based calcium indicators

Researchers wanted to develop new sensors based on fluorescent proteins because

aequorin can be used only with cell populations because it emits only a few photons, we

cannot appreciate differences in calcium concentration in single cells because I have not

enough photons emitted. This led to the generation of GFP-based calcium indicators:

the first GFP-based calcium indicators invented by Tsien and colleagues were named

CAMeleons, CAMgaroos and PeriCAMs; in all of them the molecular recognizing

molecule is calmodulin.

These three groups of calcium sensors use Calmodulin as trigger in the MRE. CaM binds

the four calcium ions with high affinity (micromolar Kd) in response to a variety of

extracellular signals that alter cellular calcium levels.

Upon Ca2+ binding the protein can interact with its target peptide M13 (M13

-Calmodulin binding domain of Myosin Light Chain Kinase). The interaction of CaM

and M13 brings about conformational changes that triggers the fluorescence emission

properties of the probe.

CAMeleons (FRET based sensor)

In cameleon, the probe designed by Tsien and co-workers, the CaM-binding peptide

M13 and CaM are fused together. The resulting protein construct is flanked at the

carboxyl and amino termini by blue and green mutants of GFP, respectively, and the

addition of Ca2+ leads to an increase in FRET.

Upon calcium binding,

calmodulin can interact with one

of its target peptides which is

M13 (M13 is the calmodulin

binding domain of a bigger protein called Myosin Light Chain Kinase). The interaction

between activated calmodulin and M13 brings to a conformational change that triggers

the fluorescence emission properties of the probe.

The use of CAMeleons was initially limited by a number of major problems:

1. Binding with calcium induced quite reduced differences in FRET efficency

2. Sensors composed of two FPs are quite large and cannot be easily be targeted to

all intracellular compartments (i.e. mitochondrial matrix)

3. pH sensitivity

→New calcium probes based on a single FP.

GFP was mutagenized in order to sensitize the chromophore to minimal changes in the

structure of the protein (CAMgaroos).

CaMgaroos

In CaMgaroos binding of Ca2+ to Calmodulin

inserted between a.a. 145 (tyr) and 146 of YFP,

induces conformational modifications to the

protein that alter the protonation of the chromophore (altering its pKa) mimicking

acidification or alkalinization events that in turn alter the emission of photons from the

FP. In Camgaroos, Ca2+ binding induces an increase in the emission of fluorescence.

Pericams

Circular Permutation: the N-terminus and

C-terminus of YFP are fused though a linker

and at a.a. Tyr 145 a new N-terminus in generated and fused with CaM. At a.a. 144, a new

C-terminus is generated and fused with CaM binding peptide M13.

Pericams are genetically encoded calcium sensors in which circular permuted YFPs

(cpYFPs) are closed between CaM e M13. Mutation in the sequence of the biosensors

generated a number of different versions of the probe.

PHARMACOLOGY

Fornasari

2 projects (private and public) for studying human genome. It took 11 years (1990-2001) to

sequence 9 genomes (4 private e 5 public). It was necessary so much time because there are a lot

of repetitive sequences → difficult to find their correct position. The final version of human

genome was released last year. Nowadays it is possible to get information about all genomice in a

few days.

They thought that sequencing the human genome would have an impact on therapeutics → drugs

with specific targets. At that time they had almost 500 drug targets, nowadays we have 600 -700

drug targets. Some targets are:

- 50% receptors → they are mostly membrane receptors. Receptors are molecules that can

be associated with membrane or intracellular, all intracellular receptors are basically

transcription factors. There are 3 different membrane receptors: channels that operate

binding a specific target, G protein