Congratulations! You have just been promoted to chief scientist (and bottle-wash
ID: 190807 • Letter: C
Question
Congratulations! You have just been promoted to chief scientist (and bottle-washer) for Hot Heads Biotech, Inc. (HHI). HHI is focused on discovery research of the transient receptor potential (TRP) family of channels. TRPs are non-selective cation channels that are found in sensory neurons and involved in temperature sensation. HHI has been approached by Nintendo to help in the development of their new virtual realty gaming system which includes their proprietary HEAT™ technology. HEAT™ is a system that allows for the controlled release of a chemical agent to the finger of the gamer which gives the sensation of touching a hot or cold object. Nintendo would like HHI to identify a series of chemical agents that can stimulate the sensation of different temperatures. Your job is to put together a project proposal describing how HHI will approach this issue.
Some notes about TRP channels:
TRP channels are thermal sensitive channels that are activated at certain threshold temperature as indicated in the chart. Activation leads to cation flow resulting in the initiation of an action potential in the neuron. There is precedent as noted in the chart for chemical agents being able to activate these channels.
HHI has cloned the series of TRP receptors listed in the chart. Your goal is to find chemical agents to stimulate the three channels for which currently no compound has been identified. You have expressed each of these channels in a host cell line. You have a library of 1,000,000 new chemical entities supplied by a combinatorial chemist. You have a fully equipped lab and have hired the other members of the class to serve as your lab slaves.
You have divided the project up as follows:
Step 1: Purify the individual channels and place into liposomes.
HOW WILL YOU DO THIS? (2 pts)
Step 2: Develop a patch clamp assay to measure channel activation.
DESCRIBE THE ASSAY? (1 pts)
Step 3: Test your library of compounds to identify channel activators.
DESCRIBE HOW YOU WILL ACOMPLISH THIS? What are your controls? How do you know if a compound is effective? (2 pts)
Mammalian thermal-sensitive channels Channel TRPV1 Threshold 243 252 230-39 227 23-28 1 7 Chemicals that evoke thermal-like response Capsaicin TRPV3 TRPV4 TRPM8 TRPA1 Group 1 TRPs Menthol, icilin Icilin, mustard oil TRPC TRPC1 TRPV TRPV1 TRPM TRPM7Extracellular TRPA TRPA1 TRPN TRPN1 TRP IntracellularExplanation / Answer
For Step 1 Liposomes are aqueous compartments surrounded by a phospholipid bilayer, which can be used to study the permeability of the membrane or to release drugs in the cells. These can be prepared by suspending a suitable lipid (such as phosphatidylcholine) in an aqueous medium. The mixture is then ultrasonically (that is, it is agitated by means of high frequency sound waves) with which a disengagement of closed vesicles of fairly uniform size is achieved. These liposomes have an approximately spherical shape, with a diameter of 50nm. Larger vesicles (up to 1 mm in diameter) can be formed by slowly evaporating an organic solvent from a suspension of phospholipids in a solvent mixture. Ions or molecules can be included in the aqueous compartment (and this is very important and will help us in step 2) of the lipid vesicles (another way of calling the liposomes) forming the liposomes in the presence of the desired substances (can be Na +, Cl- Glycine, drugs, etc.). Specific membrane proteins (in this case the TRP channels) can be solubilized in the presence of detergents and then added to phospholipids that will form the liposomes. These liposome-protein complexes (proteoliposome) are very practical tools when examining the functions of a wide variety of membrane proteins.
For step 2 The proteoliposomes are assembled with some ion that permee through the TRP channels. The temperature of the medium rises to reach the threshold temperature of the channel and the change of ion concentration in the medium is measured (that is, if there were no ions in the external medium, now after opening the channel by increasing the temperature we must find ions)
For step 3, the proteoliposome is placed at room temperature and it is tested with each compound to see if it opens the channel. If the compound works, there will be release of ions to the external medium. The positive controls would be compounds that we already know work and open the channels even at room temperature.