Cholera, Cystic Fibrosis and Hangovers G proteins and second messengers

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

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

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 ... …

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.

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

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

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

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

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

Protein kinases

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.

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

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

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

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

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

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

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)

IP3 causes calcium release from the ER

GPCR NE a a ER/SR PLC PLC IP3 IP2

So what has all this to do with cholera?

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

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

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

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

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