1. Cholera, Cystic Fibrosis and Hangovers G proteins and second messengers

3. Cholera
Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae.
The toxin released by the bacteria causes increased secretion of water in the intestine, which can produce massive diarrhea.
Various countries have weaponized cholera toxin for ChemBio warfare

4. The 1991 South American Epidemic
Incidence: 1 in 100,000 worldwide
Infection via contaminated food and water
1991 Peru Epidemic
Over 1 million cases
Nearly 10,000 fatalities

5. Cholera in the news in the past few hours!
Cholera outbreak infects over 100 students in northern Nigeria AFP - 39 minutes ago KANO, Nigeria (AFP) — At least 116 female students in northern Nigeria have been hospitalized with cholera after consuming contaminated beans, ...
Cholera kills two in Tanzania The Times, South Africa - 5 hours ago DAR ES SALAAM - Two people have died from an outbreak of cholera in southern Tanzania, a local health official said. They were among five people admitted to ...
Angola: No Cholera in 48 Hours AllAfrica.com, Washington - 8 hours ago Angola's central Huambo province capital city has not recorded any new case of cholera in the last 48 hours, apart from the 134 people affected since the ...
Two die of cholera in Murshidabad Times of India, India - 20 hours ago BEHRAMPORE: Two people have died of cholera at Beldanga over the last five days. Another 60-odd people are suffering from the disease. ...
Cholera claims a woman's life in Kliptown, Soweto Anarkismo.net - 13 hours ago One of two confirmed cases of cholera in Kliptown, Kelebogile Malefane's death has seen the Gauteng Department of Health spring into an awareness campaign ... …

6. Progression
Cholera is one of the most rapidly fatal illnesses known. A person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided.
More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days.
Severe dehydration can cause death.
Given adequate fluids, most people will make a full recovery.
Caused by cholera toxin, a G protein inhibitor.

7. Signaling molecules operate over various distances
Receptor proteins exhibit ligand-binding and effector specificity

8. Ligand solubility determines receptor location
Steroid (cytosolic) receptors
Cell surface receptors

9. Dissociation Constant
Receptor equations
Dissociation Constant
Fractional occupancy
H = hormone
R = receptor
RH = receptor:hormone complex 1
Occupancy
[H] KD

10. Conserved signaling molecules
GTPase switch proteins (G proteins)
Protein kinases
Second messengers

11. GTPase switch proteins (a.k.a. G proteins)
“Ras” is just the proper name of a particular G protein
Two roles:
Molecular switches
Molecular timers

12. Protein kinases

13. Second messenger functions
Second messengers are small molecules that convey the message from the receptor to the cell interior.
They provide for:
Amplification - many second messengers are generated per signalling event
Diffusion - most are small and diffusible, so they can go where the signaling molecule cannot.

14. The most common second messengers
Ca2+ calcium is the most common!
IP3 inositol triphosphate
DAG diacylglycerol
NO· nitric oxide
cAMP cyclic AMP

15. Second messengers may be generated by enzymes
Adenylyl cyclase makes cyclic AMP (cAMP)
cAMP is broken down by phosphodiesterase

16. G protein-coupled receptors
All G protein-coupled receptors (GPCRs) contain 7 membrane-spanning regions
GPCRs are involved in a range of signaling pathways:
light detection,
odorant detection
detection of many hormones
Detection of many neurotransmitters

17. Specificity in GPCRs
The extracellular domain determines ligand specificity
The cytoplasmic domain determines G protein specificity
Together, these two domains link a particular hormone to a particular signaling pathway

18. G protein-coupled receptors and their effectors
Many different cell-surface receptors are coupled to trimeric G proteins
So called because they actually consist of three subunits.
α, β, and γ
We’ll talk about “monomeric” G proteins later.
Ligand binding activates the receptor, …which activates the G protein, …which activates an effector enzyme …to generate an intracellular second messenger
Note: G proteins can either stimulate (Gs) or inhibit (Gi) effector enzymes

19. G proteins and cAMP Ligand binds to GPCR
Conformational change is transmitted to trimeric G protein
Nucleotide exchange, GDP leaves and GTP binds
Subunits separate
Subunits bind to and activate proteins, including “effector enzymes”
Second messengers are typically activated
The alpha subunit hydrolyzes GTP to GTP
The complex of subunits reforms and inactivates

20. Inositol triphosphate (IP3)
Another second messenger
Generated by the effector enzyme phospholipase C
PLC cleaves phosphatidyl inositol bis-phosphate (PIP2) in the membrane into IP3 and diacyl glycerol (DAG)

21. IP3 causes calcium release from the ER

22. GPCR NE a a
ER/SR
PLC
PLC
IP3
IP2

23. So what has all this to do with cholera?

24. Na+ K+ H2O Cl- P CFTR Na+ Basolateral Cl- Na+ Apical Na+ K+ H2O
As starting conditions, assume that the CFTR is closed, that sodium is low in the airway mucus, as is chloride.
Phosphorylation opens the CFTR in response to hormonal signals
Chloride flows out of the cell onto the apical surface
This helps retain sodium on that surface
The combination is osmotically active, drawing water out of the body into the mucus
The mucus is diluted and less viscous
Na+
Na+ K+ K+
Apical
H2O
H2O

25. H2O Cl- GPCR CFTR VIP a a P Na+ AC AC Basolateral Cl- Na+ cAMP Apical
PKA
cAMP
Na+
cAMP AC AC
PKA
Apical
ATP
H2O
H2O

26. Cholera toxin
Cholera toxin ADP-ribosylates the Gα subunit of the G protein that activates adenylyl cyclase
Gα can no longer hydrolyze GTP

27. In Cholera… Cl- Na+ H2O Cl- GPCR CFTR P Na+ R a AC AC Basolateral
ADP-ribosylation P
PKA
cAMP
PKA
cAMP
Na+
Na+ a R
cAMP AC AC
PKA
Apical
ATP
H2O
H2O

28. Quick fix for cholera and hangovers
A simple treatment for cholera, or ANY dehydration, is to mix half a teaspoon of table salt and 8 teaspoons of sugar in 1 quart of water.
= lots of water + small amounts of salt (NaCl) + glucose (sucrose = glucose-fructose disaccharide).
Sodium drives the transport of glucose into the epithelial cell
Glucose is osmotically active, and trapped in the body, thus helping retain water.
Gatoraid works too! P
Basolateral
Cl-
CFTR
Na+
Na+ K+
glucose
H2O
H2O