alcohol

So if you’ve ever been to a bar, then I’m sure you’d agree with me in saying “it’s a world of its own”. That place might as well be a faraway land, filled with mystical creatures because it’s like no other place on earth. Social norms are thrown out the window; here it is socially acceptable to become overly intoxicated, fist pump or to wear clothes that would make your mother cry. People do things they would never imagine themselves doing. I once saw a newly divorced man in his 40’s transform into a “bro” before my eyes. Within an hour of showing up by himself, he was taking body-shots off of girls and buying rounds for me and my friends. Sometimes you leave the bar scarred for life. There have been numerous occasions where I’ve left wishing I could wash my eyes with hydrochloric acid to un-see the sights from the night. The bar scene also has a hierarchy completely separate from the rest of the world. You can have a testosterone filled burger-flipper outmatch a powerful CEO or watch as flocks of men bow to the every command of a philosophy-major freshman. The bar is a strange machine in which alcohol is the oil. Next time you go to the bar, (if it’s not your scene then ask a friend) look around and observe alcohol’s variable effects on those around you. It can drastically change from person to person. This difference makes me wonder if it is simply how each individual perceives their experience or if there is actually some biological variance between fellow “party-rockers”. Is there some genetic predisposition which is driving this variability?

Funny enough, researchers at the University of San Francisco were asking the same question. Their main research is centered around alcohol’s effect on the body and trying to map genes related to alcohol use disorders (AUD). The third highest cause of premature death in Canada is due to AUD, so it’s kind of a big deal. I assume if you’re reading this you know that the small molecule which leads to alcohol’s effects on the body is ethanol. This little guy is hard to study in a full biological system because of its wide array of effects it can have on many different parts of the human body. If you read my last epic blog, then you know of ethanol’s effects on cognitive behavior due largely to its ability to act on the GABA receptors in the brain.

These hard-working researchers in San Francisco wanted to find another pathway affected by ethanol and possibly a genetic link to ethanol’s effects. It was time for our friendly neighborhood model organism to come to our aid, Drosophila melanogaster. First, they inserted a bunch of P-elements randomly into a group of fruit flies. After that, they started pumping them full of alcohol like some frat boy doing a keg-stand. I’ve got to say, there is a scary resemblance between a drunk fly and us (which is probably why flies were chosen). If you’re as curious as I was to see what drunken fly looks like, check out the link below, you’re welcome.

Oddly enough, they noticed one fly that just stood there and took alcohol’s abuse without passing out. This single majestic fly was one of a kind and caught the attention of the researchers…and was soon ground up so its genome could be sequenced. They found that the P-element was inserted into a known gene. After extensive intellectual debate over what to name this gene, they decided on “happy hour” (I kid you not). So once that was settled, they used qualitative PCR to measure how the expression of the gene was altered by the P-element insertion. The happyhour (hppy) gene mutant was only being expressed by half of the wild-type gene.

To check that it was indeed the hyyp mutation which resulted in this alcohol resistance superpower, they inserted the wild-type gene into the P-element mutant fly and showed that these flies passed out at the same time as the everyday normal fly, no more superpowers. Using GAL4/UAS insertions into the fly genome, they were able to test several cell-type specific promoters to pin-point the hppy’s effects of alcohol resistance. The elav promoter is only turned-on in neuronal cells and was linked to hppy mutations and ethanol sensitivity. So now the plot thickens.

Previous studies on the epidermal growth factor receptor/extracellular signal-regulated kinase (EGFR/ERK) pathway revealed there might be a connection to ethanol sensitivity. Sure enough, when the researchers over-expressed some of the players in this pathway and forced-fed the flies with alcohol, they also saw a resistance, much like the hppy mutants. Now the question was: “where does hppy function in this pathway”? They set down their beers for the next experiment and moved to a more studied EGFR pathway, fly eye development. By over-expressing hppy they found no change in eye development. Once the EGFR was over-expressed, the well documented “rough-eye” phenotype was seen. They then over-expressed both hppy and EGFR and noticed a decrease in the roughness of the eye and therefore concluded hppy represses the pathway. When hppy was ever-expressed with a constitutionally on EGFR, no change in eye development was seen, supporting the notion that hppy acts upstream EGFR.

Changes in fly eye development

Changes in fly eye development

Finally, the researchers moved from alcohol, to eyes, to drugs (but not of the party variety). Two FDA-approved drugs known to inhibit EGFR, Erlotinib and Gefitinig, were utilized. These drugs, like hppy, repress the EGFR pathway. As these clever scientists predicted, the drugs made the fruit flies more sensitive to alcohol. Acting much like a 16 year old girl after her first Bacardi Breezer, they immediately passed out after even small doses of alcohol, once fed the drugs.

You can tell this is when the scientists started getting cocky because they then moved on to mice. To their surprise, the mice reacted the same way to the drugs and alcohol as the flies. The little mice experienced their little blackouts earlier and woke up with their little hangovers (cute, right?). The final experiment gave the mice the choice between water, sugar-water and alcohol (no-brainer). Before they administered the drugs they were sucking back the liquor like Jim Lahey. After giving the mice the EGFR inhibitors, their preference for booze drastically decreased. So not only did they show physiological changed and passed out quicker, but they showed behavioral changed and a drastic decrease in preference for alcohol.

effects of alcohol

Effects of alcohol on mice given EGFR inhibitors

These scientists were able to find a genetic link and describe a new pathway involved in ethanol sensitivity, and potentially find a suitable cure for AUD with two approved drugs. Meanwhile, I can barely cook Kraft Dinner without burning it. As research continues in this field, we see more and more how a person’s genetic makeup can affect their response to alcohol. So next time you see someone shirtless and dancing on the bar maybe you’ll think to yourself “poor guy, it’s not their fault, they were born that way”.

 

  • This awesome paper mentioned above- Corl, A. B., Berger, K. H., Ophir-Shohat, G., Gesch, J., Simms, J. A., Bartlett, S. E., & Heberlein, U. (January 01, 2009). Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors. Cell, 1
  • Fly getting “white girl wasted”- http://www.youtube.com/watch?v=ZCJi8nIGd-s

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Let’s set the scene, I just graduated from high school, I was 18 and shipped off to Quebec City for the summer to finish my Navy training. The whole summer basically became an Erlenmeyer flask filled with liquor, excessive self-worth and poor life choices. Not counting boot-camp, where we were treated worse than pound puppies set to Sarah MaLachlan music, this was my first summer away from home where I was free to do as I wished. I didn’t know it at the time, but during that summer I performed more biology and chemistry inside my body than I’d ever do in all four years of undergrad labs.

One of my craziest escapades of the summer occurred on the day of our first exam. We sat down at nine to write and by noon we were back in our rooms and ready to celebrate. Now, what do four sailors with excess money and a lack of social etiquette do to celebrate during the afternoon? Order in a pizza and relax? Wrong! We cracked a bottle of Jägermeister and started throwing Jäger bombs down our throats. As soon as the liquor reached my stomach, the ethanol started doing its work on my cognitive abilities.

Ethanol is a very small and soluble molecule which allows it to be easily absorbed into the blood stream. Studies show that about 20 percent of the ethanol is absorbed into the stomach. Alcohol is also shown to easily pass the blood-brain barrier. Previously, it was thought that alcohol acted non-selectively in the brain by disrupting the lipid bilayer of the neuron cells. This disruption would allow the leakage of ions and the result would be less excitable neurons. It was not until recently that this theory was disproven. Alcohol actually targets ligand-gated ion channels. Some channels are inhibited by ethanol’s effect and others are induced. A hotbed of research over the last few years has focused on alcohol’s effect on one of the primary inhibitory neurotransmitters in the human brain, γ-aminobutyric acid type A channels (GABA). As you will soon see, these channels are very important in everyday function.

Almost as soon as we started drinking, we got the bright idea to hop on our bikes and head to the outdoor public pool to cool off. We threw a case of beer into our backpacks and made the 20 minute trip without a hitch. Once there, the Jäger must have made it into my stomach where studies show the other 80 percent of absorption occurs. This large intake of ethanol began to influence the GABA receptors on my neurons. In a normal cell, GABA is released into the synapse and binds to the GABA receptors.  This allows chlorine ions to enter the post synaptic neuron. These negative ions make the neuron less excitable. The GABA receptors are pentamers with a high homology to nicotinic receptors. There are many subunits which can make up a GABA receptor and this leads to a rich pharmacology depending on which subunits are present. The typical subunit has an extracellular N-terminus with a ligand binding site and four membrane spanning regions. Alcohol also binds to the GABA receptor and allows it to stay open for a longer period of time which results in more chloride ions passing into the neuron. The influx of chloride ions moves the postsynaptic membrane potential farther from the threshold and from firing. An interesting study conducted in 1992 shows that the N-terminal and loops is the site of ligand binding, but there are also other distant sites on the protein which can bind a number of compounds that allosterically modulate the neurotransmitter. This distant site binds many compounds such as benzodiazepines, anesthetics and of course ethanol. The binding to the distant site is only able to activate the channel in the presence of GABA bound to the receptor. There must have been plenty of this duel binding occurring at that time because we were starting to really feel the effects of our actions.

JJ1

JJ2

 

 

 

 

 

 

 

Unfortunately for me and my friends, the first brain center to be affected is the cerebral cortex. Right before heading into the pool area, we shot-gunned a couple beers, threw on our swimsuits, and then stashed our backpacks in a nearby bush. Now, being in a public setting like a pool and having an inhibited cerebral cortex can be a problem. It is responsible for the thought process and in the presence of alcohol leads to depressed behavioural inhibitory center which basically makes you think you’re a god walking amongst mere mortals. After about the fifth cannonball in a row (with a large announcement upon each attempt), the lifeguards made the call that we were unfit to remain in the pool and kicked us out. I could attribute this failure to my own misjudgment, but I would rather just blame the GABA receptors. When alcohol and GABA are both bound to the GABA receptor, the probability of the channel opening is greatly increased. The result is hyperpolarisation of the postsynaptic membrane, compared to GABA alone. Studies done on rat brains show the increase of opening due to ethanol can be as high as 260%. Ethanol induction was seen to increase channel burst frequency and burst duration as well as decrease time spent in the closed state. Now, let us not forget, opening of the GABA receptor leads to less firing of that neuron, and drunkenness.

By the time we figured out what had happened we were outside the chain fence looking at all the happy people staying cool on a hot day. I’m guessing the pre-pool beers were starting to work their wonders by this point because our limbic system was declining fast. The limbic system is the area of the brain which consists of the hippocampus and the septal area which control memory and emotions. We could not for the life of us remember where we put our backpacks. The leading theory was that we left them in the locker-room, the one which we were being refused access to. In our exaggerated state of emotion this only aided in further frustration. We soon decided to cut our losses and return back to our rooms. At that moment we clearly did not have the patience the scientists had while researching alcohol’s effects on GABA receptors. One research group wanted to find support for alcohol being involved in protein binding instead of the lipid integration theory. They tested a number of alcohols with different carbon chains and found that the GABA receptor could accommodate alcohols of up to 12 carbons but no more. This supports the theory of a binding pocket for alcohol with a defined cut-off size located on the GABA receptor. If only I knew what “cut-off” meant.

The bike ride back was much more problematic then before. First of all, I was only wearing a soaking pair of swim trunks and a helmet. Secondly, the last bit of alcohol was running rampant in my cerebellum. The cerebellum, it is responsible for everything you need to ride a bike: balance and fine muscle movement. With this key part of the brain being disrupted, the journey home was a difficult one. What came as second nature was now excruciatingly challenging. With all the focus I could muster I was able to make it home with only a minor wipeout.  I had safely landed at my destination, but the researchers at the University of Chicago wanted to see where alcohol lands on the GABA receptor. Through a mutagenesis study, they identified a single amino acid at position 267 located at transmembrane 2 of the α1 subunit which plays a role in alcohol binding. If replaced by a small serine group, the size cut-off for the alcohol carbon chain was increased (larger pocket). In contrast, if replaced by a large amino acid, the size cut-off was decreased (smaller pocket). To think, they discovered all that and I was just happy to get back.

When most people return home they would do the sensible thing, like chug a glass of water and pass out, but I am not “most people”. I felt like I just won the Tour de France, which required some celebration. A handful of Jäger bombs later and I was re-energized and ready to hit the clubs. The crew and I suited up and we were off to catch the bus and head to our favorite night spot. Once I left the bus I realized that if actions were not immediately taken, there’s a good chance my bladder was going to explode. You see, one of the last places alcohol seeps into is the pituitary gland. Here it inhibits the gland from releasing anti-diuretic hormone. This hormone is an important target to the kidneys which allow the kidneys to reabsorb water. Without this reabsorption, the kidneys produce an excess of urine. I panicked and sprinted to the nearest bush.  Too bad this bush happened to be on the front yard of the Quebec parliament. Doesn’t matter, mission accomplished.

By this time, I really could of used the help from the scientists who designed an allosteric modulator of the GABA receptor, imidazobenzodiazepine Ro 15-4513 (or Ro for short).  This drug has been shown to actually reverse the intoxication effects of ethanol in lab rats. This compound binds to several types of subunits of the GABA receptor and has an affinity of about 10 times greater than ethanol. This allows Ro to out-compete ethanol for the binding sites. The trick is that Ro does not activate the GABA receptor to the same extent as ethanol, so the effects are lessened.

I’d like to say this story ends with an epic ending and a crazy end to a crazier day, but it doesn’t. By the time we all reached the four story club the ethanol in our systems had reached its final destination, the medulla. This part of brain regulates all the really important stuff that keeps us alive like temperature, breathing, heart rate and consciousness.  The bouncer could clearly see that we were in no state to be “party rockin” and turned us away. Shunned from our final destination, we finished off the night in classic Quebec fashion, poutines. I’m sure you’re wondering “wow, how did YOU feel the next morning?” but I’m going to save that for another blog post. Hope you all enjoyed this little story and most importantly, I hope you learnt about alcohol’s effects on GABA receptors. Thanks for reading and stay tuned for another post filled with drunken debauches, science and shameless fun!

The studies discussed in this post can be found here. I recommend giving them a read:

1)      Davies M. The role of GABAA receptors in mediating the effects of alcohol in the central nervous system. J Psychiatry Neurosci. 2003 Jul;28(4):263-74. Review. PubMed PMID: 12921221; PubMed Central PMCID: PMC165791.

2)      Suzdak PD, Glowa JR, Crawley JN, Schwartz RD, Skolnick P, Paul SM. A selective imidazobenzodiazepine antagonist of ethanol in the rat. Science. 1986 Dec 5;234(4781):1243-7. PubMed PMID: 3022383.

3)      Nakahiro M, Arakawa O, Nishimura T, Narahashi T. Potentiation of GABA-induced Cl- current by a series of n-alcohols disappears at a cutoff point of a longer-chain n-alcohol in rat dorsal root ganglion neurons. Neurosci Lett. 1996 Feb 23;205(2):127-30. PubMed PMID: 8907333.

4)      Li D, Sulovari A, Cheng C, Zhao H, Kranzler HR, Gelernter J. Association of Gamma-Aminobutyric Acid A Receptor α2 Gene (GABRA2) with Alcohol Use Disorder. Neuropsychopharmacology. 2014 Mar;39(4):907-18. doi: 10.1038/npp.2013.291. Epub2013 Oct 18. PubMed PMID: 24136292; PubMed Central PMCID: PMC3924525.

5)      Ye Q, Koltchine VV, Mihic SJ, Mascia MP, Wick MJ, Finn SE, Harrison NL, Harris RA. Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position alpha267. J Biol Chem. 1998 Feb 6;273(6):3314-9. PubMed PMID: 9452448.

6)      Suzdak PD, Paul SM. Ethanol stimulates GABA receptor-mediated Cl- ion flux in vitro: possible relationship to the anxiolytic and intoxicating actions of alcohol. Psychopharmacol Bull. 1987;23(3):445-51. PubMed PMID: 2893423.

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Image  —  Posted: February 22, 2014 in Uncategorized

First post!

Posted: February 13, 2014 in Uncategorized

Alrighty folks, I’m going to keep this one short and sweet. I heard a great joke today and it would be a sin not to share it with you lovely people, “according to most chemists: alcohol is not a problem, it’s a solution”! That’s all for now readers.

Happy Thursday!