Download Discussion_5_Paper_Questions

Paper Title: “An SCN9A channelopathy causes congenital inability to experience
pain” Cox, 2006.
http://www.nature.com/nature/journal/v444/n7121/abs/nature05413.html
Purpose: The goal of this exercise is to (1) introduce students to the structure of
scientific papers & (2) evidence of neurobiological research that is relevant to
material they have just learned about regarding the electrophysiology of neurons
and its clinical impact.
Activity 1: Discuss what the purpose of each of these sections is.
The structure of this paper is organized into:
1.
2.
3.
4.
5.
6.
Abstract
Introduction
Results
Discussion/Conclusion
Methods
Supplemental Data
An overview of Nav1.7:
SCN9a encodes the alpha subunit of the Nav1.7 voltage-gated Na+ channel.
Together with SCN1b & SCN2b, these create a functional Nav1.7 channel. Nav1.7 is
expressed in 2 tissues, nociceptive DRG neurons and sympathetic ganglion neurons.
For the detection of pain, the nociceptive DRG neurons are essential.
The Nav1.7 gene functions as an “amplifier” of nociception. Nociception is the
detection/perception of pain, and is activated by nociceptors in nerve fibers.
Nociceptors are a type of sensory receptor which responds to pain. In response to
pain, nociceptors produce “generator potentials.” In this case, they are small voltage
depolarizations across the neuronal membrane in DRG neurons. When there are
small depolarizations from nociceptors, the Nav1.7 channel amplifies the initial
generator potential, further depolarizing the neuron. If enough depolarization is
reached, an AP is initiated. To clarify, the Nav1.7 does not detect the painful stimuli,
it amplifies the initial detection, thus an “amplifier.”
Activity 2: Questions to guide student understanding of the substance in the paper:
1. In the paper, the phenotype is an insensitivity to pain. Contemplate the “plan
of attack” researchers pursued to hone in on the genetic mutations
responsible for the phenotype.
a. The human genome consists of billions of DNA base pairs. How can a
researcher localize the mutation to a smaller region of DNA (say, to a
region that is 10^6 bp, rather than 10^12)?
2. In the paper, researchers wished to examine the functional effects of the
candidate mutations they identified as likely causes for the insensitivity to
pain. To do this, the researchers used cell culture techniques, which involve
injecting foreign DNA (plasmids) into cells growing on plates. In this paper,
researchers use 2 plasmids.
a. Why does the SCN9a plasmid include DsRed9 & the SCN1b/SCN2b
plasmid include EGFP? Worded another way, what is the purpose of
including the genes DsRed9 and EGFP on each plasmid? (reference
figure 4a, 4b)
b. Why, in one set of experiments, do the researchers transfect (i.e. inject
DNA into cells) only the SCN1b/SCN2b plasmid into the HEK293 cells
without the SCN9a?
3. Figure 4c represents the current response of a voltage clamp experiment.
First, define the experimental groups. Second, explain the purpose of the
experiment and the response difference between wild-type and mutants in
the context of the voltage clamp experiment.
4. Figure 4d represents the current response of a voltage experiment. In Figure
4c, the voltage clamp duration is 50 ms, whereas the duration for the
experiment in Figure 4d is for 500 ms. This difference is related to “voltage
dependence of steady-state inactivation.” First, define the experimental
group the researchers are looking at. Second, explain the chart on the right in
figure 4d.
A final note: Consider the knowledge base required to conduct the research presented
in this paper. This includes extensive knowledge in: bioinformatics, genetics,
electrophysiology, neuroanatomy, molecular biology.