Download M TO O WEEK ONE READING IN PHYSIOLOGY

BIOLOGY PORTION
SCIENCE OF SENSORY PERCEPTION FALL 2015
WEEK TWO WORKSHOP: TOUCH
Voltage-Gated Sodium Channel in Grasshopper Mice Defends Against Bark
Scorpion Toxin
Ashlee H. Rowe,1*†‡ Yucheng Xiao,2† Matthew P. Rowe,3§
Theodore R. Cummins,2 Harold H. Zakon1,4
The workshop this week will allow us to seminar on some advanced reading in the field
of cell physiology and molecular biology as it relates to pain. I chose this particular
article because I thought it was interesting and instructive. It is also difficult, particularly
for those who are not used to reading primary literature. Because of this, I have tried to
structure questions that will guide you through the paper. You need to be persistent and
patient when reading primary scientific literature. You need to take notes on a separate
piece of paper and/or the margins of the readings. You need to look up terms that you do
not understand. You need to understand figures. Some of this paper is “impenetrable”
for readers who are not in this research field. I will try to help you differentiate what you
should understand from this paper versus what you can ignore, through these guided
questions. A reasonable time to spend on this is four hours.
Please do the readings and then come to class prepared to answer these questions with
your fellow students. I EXPECT THAT YOU WILL BE ALREADY FAMILIAR WITH
THE PAPER, AND THAT YOU HAVE SPECIFIC QUESTIONS FOR ME AND
YOUR COLLEAGUES. DO NOT COME TO WORKSHOP WITHOUT READING
AND ATTEMPTING TO ANSWER THESE QUESTIONS BEFOREHAND.
1. Where was this paper published and when?
2. Read through the abstract quickly and try to keep track of a few terms that you
will look up later.
a. What is Nav1.7 and Nav1.8? What is the difference between them?
b. Which one is a “non target” for the toxin in most mammals?
c. Make a table with the following categories: Nav1. and Nav1.8, domestic
house mouse and grasshopper mouse. Now fill in the table with the
following: bark scorpion venom binds and causes pain, bark scorpion
venom doesn’t bind, and bark scorpion venom binds and inhibits pain.
3. What does “adaptive function” mean?
4. What are the advantages of having a pain response? Why might this be an
adaptive trait (from an evolutionary perspective)?
5. Keep track of the “characters” and their Latin names in this paper by writing them
down (bark scorpion, grasshopper mouse, domestic mouse). Now look them up
on google images so you have a sense of what they look like (I did!)
6. What does “sympatric” mean?
7. What is an “amino acid variant”?
8. Look at figure one. In your own words, describe the experiment
a. What variable did they measure?
b. What was the negative control?
c. What was the positive control and why did they use it? (figure 1b)
9. “Effects of Venom on Dorsal Root Ganglion–Expressed TTX-R Na+ Current”.
This is a more difficult section of the paper.
a. Review what a voltage gated sodium channel is and how on a molecular
basis, a change in voltage along a plasma membrane can make a change in
the conformation of a transmembrane protein channel that can open or
close it for ions to move into a cell.
b. Note that these dorsal root ganglion have two voltage gated sodium
channels on their cell membrane.
c. How did they use TTX to confirm that bark scorpion venom toxin does
not bind to Nav1.8 and activate the neuron in the domestic house mouse?
In other words, how did they isolate the effect of the venom on Nav1.8 on
grasshopper mouse compared to domestic mouse? Add TTX-S and TTXR to your table!
d. The details of this experiment are tricky. Essentially they measured the
amount of sodium entering (current) the dorsal root ganglion cell through
Nav1.8 in grasshopper mice and house mice with and without venom.
What were there findings?
10. “Effects of Venom on Nociceptor Excitability and Action Potential Generation”
a. Remember that these neurons have two sodium voltage gated channels:
Nav1.7 and Nav1.8. Which one is responsible for initiation of action
potentials, and which one is responsible for sustaining action potentials
through propagation? Add this to your table!
b. They essentially delivered a small voltage to initiate action potentials, and
then measured action potential frequency to see how they were sustained.
They then compared this frequency between grasshopper mouse and
domestic house mouse with and without venom. What did they find? Be
able to interpret figure 3a and 3b.
c. Why did these findings prompt them to pursue further work? (see below)
11. “Effects of Venom on Response to Alternative Painful Stimuli”
a. What was the “alternative painful stimuli”?
b. Interpret Figure 3c
12. The rest of the paper goes through the molecular characterization of how the
venom interacts with Nav1.8 in grasshopper mice. Here are some things you
should review in order to understand this part:
a. What is a transmembrane protein?
b. What is a protein domain?
c. What is a hydrophobic and hydrophilic amino acid? Where would they be
in relationship to a phospholipid bilayer?
d. What is a charged amino acid residue? Is this hydrophobic or
hydrophilic? How would this interact in voltage dependent movement of
a protein?
e. Why would the researchers even compare the molecular structure of these
proteins isolated from domestic versus grasshopper mice? How did they
do it (molecular technique)?
f. How many domains are in the Nav1.8 protein? Which one does the
venom bind to?
g. What is a protein chimera?
h. What type of in vitro system did the researchers use to test the effects of
changing specific domains of the protein? Interpret figure 4b
i. What happened when they replaced otNav1.8 domain II, with mNav1.8
domain II? Which part of figure 4 shows this?
j. Why did the researchers use site directed mutagenesis for?
k. What do the letters stand for in Figure 5a?
l. What were there findings in the site directed mutagenesis work?
13. How did they determine whether the amino acids critical for venom-pain
insensitivity are unique to grasshopper mice? Why did they do this? Were you
surprised by the results?
14. What does “counteradaptation” mean?
15. So do grasshopper mice feel less general pain than other mice? Why is the
answer to this question important?
16. Compare and contrast grasshopper mice to naked mole rats in terms of which
channel has changed, and what they are protected against.
17. What is meant by “co-evolution and arms races at a molecular and biochemical
level?”
18. How might this research actually help people?