The Versatile GABAa Chloride Channel Receptor Complex

In today’s industrialized society we are constantly exposed to work related stresses. Consequently, anxiety and insomnia (sleeplessness) have become quite common. No wonder, we are using anxiolytics and sedatives more often; to get relief from the anxiety and insomnia respectively.

Benzodiazepines such as diazepam (Valium), chlordiazepoxide (Librium) can effectively treat anxiety and insomnia. They do so by binding with a receptor (called Benzodiazepine-GABAa-chloride ion channel complex [henceforth to be referred to simply as GABAa receptor]) in nerve cell membranes. It is known that most drugs (medicines) exert their actions by combining with receptors: macromolecular complexes present in the cell membrane or within the cytosol or the nucleus.

The GABAa receptor is a very versatile receptor complex (a hypothetical model is shown at the bottom). Its main action is to inhibit transmission along neurons in which they are present. Normally, proper functioning of the brain is ensured by a balance between the action of excitatory and inhibitory neurotransmitters [henceforth to be referred to simply as NTs]. Simply put, excitatory NTs (for example, glutamate) give a green or go signal; while inhibitory NTs (such as GABA) tell the nerve not to fire (red or stop signal). In this connection, it must be said that GABA (gamma amino butyric acid) is the most important inhibitory NT. When GABA binds with the GABA receptor ionophore complex, the receptor changes shape (conformation); and then a centrally located chloride channel, that is a part of the receptor itself, opens. Since the concentration of the chloride ions (Cl-) is much more on the outside of the cell than on the inside, Cl- now rushes in due to the increase in chloride conductance. The cell voltage goes further down and the interior of the cell becomes more negative (hyperpolarized) with respect to the outside. The cell becomes less excitable and is thus inhibited.

Apart from maintaining the much needed critical balance already mentioned, they also ensure that the brain works in a relatively noise free environment. Billions of neuronal units are always firing in the background creating a constant ‘noise’. A constant release of GABA by the brain drowns out this noise thus improving the ‘signal to noise’ ratio, making the brain’s task of finding the proverbial ‘needle in a haystack’ a lot easier.

The GABAa receptor not only binds with GABA, but it is a binding site for various other ligands. But before we discuss them, let us briefly analyze its structure first. The receptor has a pentameric structure which means that it consists of five subunits, and each subunit has four membrane-spanning (transmembrane) domains (see picture). And there are many of the polypeptide subunits to choose from a vast array consisting of alpha, beta, gamma, delta, pi, rho and so on. (In addition, there are six different forms of alpha, 4 beta and 3 gamma subunits). Thus, it’s no wonder that a great variety of GABAa receptors will be found, given the possible permutations!

This receptor heterogeneity explains actions of various pharmaceuticals on the receptor. One major form of GABAa receptor (found throughout the brain) consists of two alpha1, two beta2 and one gamma2 subunits. In this isoform, GABA ‘somewhere’ between alpha1 and beta2 subunits, and benzodiazepines bind with the BZ1 (also called omega1) pockets located between alpha1 and gamma2 subunits. Benzodiazepines act only when the receptor isoform has one of the following alpha subunits: 1, 2, 3 or 5 and the subunit should have a conserved histidine residue in the N-terminal domain. In ‘knock-in’ mice where histidine has been replaced by arginine in the alpha1 subunit (alpha1H101R; H for histidine and R for arginine in the 101st residue of alpha1 subunit) there was no sedation or amnesia (as evidenced by their unchanged ‘energy’ and memory to electric shocks). It may be mentioned at this moment that the so called ‘date rape’ pills exploit the amnestic properties of benzodiazepines. The drug plays tricks with the victims’ memories. However, the anxiolytic and muscle relaxant properties were retained in these mice.

These mice also do not respond to the hypnotic effects of zolpidem and zaleplon, non-benzodiazepines that act at GABAa receptors containing alpha1 subunits. But in mice with selective histidine arginine mutation in the alpha2 subunit of GABAa receptors, resistance to the antianxiety action of benzodiazepines has been seen. Based on these observations, it is thought that alpha1 subunit mediates sedative and amnestic effects, while alpha2 takes care of the anxiolytic and muscle relaxant ones. It also seems that we are poised to make better benzodiazepines in future (like one that works in anxiety but doesn’t wreak the patients’ memory).

Lastly, the versatility. The GABAa receptor also binds barbiturates (urea derivatives used as anesthetics, anticonvulsants, Marilyn Monroe supposedly died of its overdose) in addition to the benzos. Alcohol, alphaxolone (a steroid anesthetic), etomidate (a short acting anesthetic), propofol (diprivan, Michael Jackson supposedly used it), volatile anesthetics like halothane, anticonvulsants like gabapentin and vigabatrin, anthelminthics like ivermectin, and neurosteroids (metabolites of androgen and progesterone) exert part or all of their actions by acting through this receptor, thereby hyperpolarizing the neuron. Conversely, convulsants picrotoxin blocks the chloride channel directly, while bicuculline blocks the receptor’s GABAa binding site causing depolarization and convulsion. There's a lot more than this mere exegesis, and I hope to discuss about it furher later.

Last modified: never
Reference: Bertram G. Katzung, Basic and Clinical Pharmacology, ninth edition
Pharmacology: Rang, Dale, Ritter, Moore
Wisden, W., & Stephens, D. (1999). Pharmacology: Towards better benzodiazepines Nature, 401 (6755), 751-752 DOI: 10.1038/44482

When Bullets Lose Their Magic

German chemist Paul Ehrlich coined the term 'magic bullets' (he also used the term chemotherapy), in connection with the dyes used to visualize histology samples. He assumed that if these dyes could specifically stain the pathogens then these substances could also be used to deliver toxins that would specifically kill the rogue cells. Based on these assumptions, he developed Salvarsan (arsphenamine), a drug used in syphilis.

Medicines do not always act so magically. Sometimes medicines end up doing just the same thing it was made to cure. Such boomeranging is not uncommon. Consider the case of aspirin (acetyl salicylic acid). It is used in the treatment of fever and inflammation, among others. It inhibits the enzyme cyclo-oxygenase (COX 1 &2), thereby reducing the production of prostaglandins which works on the hypothalamus to bring about fever. Thus it acts as an antipyretic. However, in higher doses, it causes fever. This occurs as uncoupling of oxidative phosphorylation occurs at higher doses, liberating free energy in the form of heat. Oxidative phosphorylation is normally associated with the formation of ATP (high energy phosphates, the energy currency of the cells).

In cancer chemotherapy drugs used to treat cancer (usually given to lessen the tumor burden, these drugs rarely cure cancer) often cause cancer themselves. Nitrogen mustards such as lomustine (or carmustine) are examples of such antimalignancy drugs. Historically, sulfur mustard was used on the British by Germans in 1917, in a chemical warfare. It caused blisters on the skin and mucus membranes and bone marrow suppression. Its effectiveness in cancer was discovered later, nitrogen mustards came in. These agents are alkylating since they form highly reacting immonium ions in neutral or alkaline water. These immonium ions being nucleophilic, attack the DNA of the malignant cells and cross links them. The cell replication stops; the reckless and relentless progress of cancer growth is tamed.

But the most heart rendering of these are, funnily enough, the medicines for the heart themselves. Nitrates are used in angina pectoris, a medical term for chest pain or discomfort due to coronary heart disease. These compounds liberate nitric oxide or EDRF(Endothelium Derived Relaxing Factor) in our bodies, which then increase the amount of one intracellular compound called cyclic GMP (cGMP). cGMP acts on the vascular smooth muscle cells and these relax. This vasodilation brings relief in painful occlusive (spastic, atherothrombotic) coronary heart diseases. (vasodilation, incidentally, is also the basic principle of action of Sildenafil citrate, known popularly as viagra). But nitrates (polyol esters of nitric acid) such as Nitroglycerine (known variously as NTG, GTN or NG) are explosives too. Alfred Nobel found a way to stabilize this, in his discovery dynamite. Another highly explosive antianginal drug is Pentaerythrityl tetranitrate or PETN (also spelled Pentaerythritol tetranitrate). This drug (or substance!), a terrorist's pet, is often used in the making of Semtex, a deadly plastic explosive which is very tough to detect. Thus it appears that what appears life saving may be a killer too. By the way, you can safely touch (or kiss) a person who is on PETN or GTN. He (she) won't blow up, I can assure you this.

A Vaccine To Crack The Cocaine Bugs

Years ago I heard the song "Snowblind" by our one and only Ozzy Osbourne, from his album Speak of the Devil. The meaning of the song is two-fold. Firstly, people in a snowy landscape face such intense reflection of sunlight, that they might go blind. They wear special goggles to restrict sunlight, so that it doesn't burn the macular portion of the retina (the part where light rays are usually focussed.)

Another interpretation of this song could be this that someone gets blinded by the actions of cocaine (also known as snow or crack). That's precisely what Ozzy meant.

Cocaine, an alkaloid obtained from the leaves of Erythroxylum coca, produces euphoria (a feeling of elation) by increasing the amount of dopamine in the brain. Dopamine is normally liberated in brain structures such as the basal ganglia. After it has been liberated and it has finished its action, residual dopamine in the synapse is 'cleared' by dopamine transporters in a process called reuptake. These transporter proteins packs these chemicals back into where it came from (into the axon, as is shown in the picture), hence re-uptake. Cocaine binds with these transporters in such a tight embrace that these transporters fail to function: that is, cocaine functions as dopamine reuptake inhibitor. Dopamine, the pleasure (reward) chemical, which now accumulates in the synapse, gives the person a good kick. Frequent use of it through inhalation or injection (as these routes lead to faster absorption) gives them the feeling of an orgasm, which users refer to as 'rush'. Nucleus Accumbens, an area in the brain, supposedly 'reinforces' this addiction.

So isn't there a way out? Will they have to go on scratching their skin (cocaine produces a typical dermal itching sensation, Magnan sign or the cocaine bugs, where the patient feels as if insects are crawling under their skin)? Scientists are now on the verge of producing a vaccine (TA-CD Vaccine), which not only will cure their addiction but also decrease the severity of its 'withdrawal symptoms'. The human immune system can not recognize cocaine since it is a small molecule. Hence, they are combining parts of inactivated cocaine molecule with inactivated cholera proteins. After you give them a 'shot' of this vaccine, the immune system will now recognize the combine and produce antibodies against coke. Next time cocaine is consumed, the antibodies in the blood will combine with the cocaine molecule and prevent it from reaching the brain, its main seat of action. The vaccine is currently undergoing clinical trials.

Not only cocaine, vaccine is being developed against methamphetamine too (N-methyl amphetamine), another drug of abuse. The mechanism of action of the vaccine is the same. These drugs are still used in clinical practice: cocaine as an anesthetic; methamphetamine analogs, methyl phenidate (RITALIN) and pemoline are widely used in attention deficit hyperactivity disorder or ADHD. But keeping in mind their abuse potential, these drugs should be prescribed weighing the risk-benefit ratio. And when an addiction has already developed, we must cure it, may be by using a vaccine like this.

Last modified: 13 Jul 2009
Reference: hyper-links, unless specifically mentioned

The Chiral Fallacy

Do you wear your left gloves on your right hand? I know you don't. It is a fact that though the gloves are almost identical they are not interchangeable. One is the mirror image (copy) of the other. So, wearing your left gloves on your right hand is wrong (isn't left always wrong?).

When a molecule docks on another, to bind, it is said that the molecule has an affinity for it. It may (or may not) elicit a response (efficacy), as a result of this combination. But for this molecule in question; be it a hormone, an enzyme or a medicine, to combine with its consort (receptor/substrate), the molecule needs to embark on the substrate first. In a three dimensional world (leaving aside time dimension for a moment), the molecule (ligand/enzyme) has to use at least 3 points (bonds) to anchor. As shown in the figure, the pyramidal looking molecules display mirror image symmetry. Think of the black balls as indicating depth; the other three balls rest on the substrate molecule like a tripod, in a condition called 3-point attachment.

Now imagine that you are holding the black ball and placing the 'planar tripod' on three imaginary holes (of complementary colors), which correspond spatially to the three projections. No problem. You try to repeat the same thing with the other pyramid, you can't get your jigsaw done! This occurs due to the presence of an asymmetric carbon atom.

This is because though the molecules have the same molecular structures their orientations are different. This is known as isomerism, a phenomenon akin to the gloves analogy. Isomerism has immense importance in human physiology, biochemistry and pharmacology.

"Warm Ice" may help us achieve a Better Prosthesis

Lets face it. We have heard of "hot ice" and now we are discussing "warm ice", before even the cool shock of the former is yet to wear off.

Water is a fascinatingly strange molecule even though its molecular structure is apparently simple, consisting only of 1 oxygen and 2 hydrogen atoms. We will restrict ourselves with the topics relevant here, rather than digressing about the properties of water, in general.

A planet, called GJ 436, situated some 3o light years away from earth, was speculated to contain "hot ice" in it. This Neptune sized exoplanet (extra solar planet) has a surface temperature of about 300 degrees Celsius (centigrade). At this temperature, water should exist in gaseous state. But the high pressure of the planet (due to its high density), let water remain in ice form. (for example, in a pressure cooker, water remains liquid at temperatures exceeding 100 degrees Celsius, due to high pressure inside the cooker).

Exploiting a principle of biophysics, researchers (Alexander Wissner-Gross and E. Kaxiras) at Harvard University, US, have shown it by computer simulation, that by covalently bonding sodium atoms to diamond (an allotrope of carbon), some nanoscale ice would form on its surface. The sodium atoms would allow dipole interactions to occur between adjacent water molecules. A frosty layer of about 2 nm (nanometer=one billionth of a meter) would form over the surface of diamond coating. Thus, while on one hand, you can take the advantage of diamond for its 'wear and tear' resistant properties (for which it is already being used in artificial implants like heart valves, joint replacement prostheses etc.), but also we stand a better chance to ward off any possible thrombus (clot) formation by virtue of the smoothness of the warm ice. This ice is called 'warm ice' since it remains in ice form at our warm body temperature of 37 degrees Celsius. Diamond though corrosion resistant, is prone to induce or aggravate blood clotting and hence it is part of the therapeutic protocol that the patient continues taking anti clotting (blood thinning drugs: e.g. Warfarin, Ticlopidine) drugs to prevent any such calamity. Warm ice may, in future, simply make these anticoagulant drugs unnecessary.

Seems like a cool ending to a hot story!

Aspirin and Ion Trapping

The property of cell membranes or biological membranes that cover the cells all around, is an important deciding factor in permitting or denying entry of molecules to and fro. Any drug or molecule has to have an aqueous phase (water soluble) in order to dissolve in the surrounding water molecules and negotiate further. At the same time, it has to be lipid soluble, to pass through the hydrophobic domain of the cell membrane. Some peculiar instances arise from these effects.

Aspirin, a non steroidal anti inflammatory drug (NSAID), is used to treat fever, pain, coagulation disorders and various other ailments. Chemically it is a weak acid, acetyl-salicylic acid, a semi synthetic derivative of salicin, a chemical extracted from the willow bark, which was seen to be of use in curing fever and pain. When aspirin is ingested, it goes inside the stomach where the pH is in the acidic range (pH is low). Aspirin being a weak acid, can not ionize in the acidic pH of the stomach, according to Le Chatelier's principle. Thus, it behaves like a non-polar compound in the gastric lumen. Therefore, it hardly faces any difficulty to enter inside the epithelial cells, as non-polar compounds behave as lipophilic (hydrophobic) compounds. But once inside the cell, they come across an entirely different situation. The pH is high (alkaline) here. Aspirin ionizes as a result. The problem starts. Ionized aspirin now behaves as a polar compound. What a volte face! Being polar, it now can not diffuse back, as it can not cross the lipid barrier in the cell membrane. Aspirin is now trapped inside, to the detriment of the patient, as the likelihood of peptic ulceration increase. This phenomenon is known as 'ion trapping'. Thus, aspirin and other inhibitors of the cyclooxygenase pathway like ibuprofen or acetaminophen (US) [= paracetamol (UK)], plays a very important part in ulcer causation. COX enzymes are in charge of producing chemicals called prostaglandins and other bioactive molecules. Prostaglandins (PG) have a varied role spanning from fever and inflammation to pain. They also ensure the vitality and vascularity of the gastric epithelium, in addition to having an ulcer prevention role in the stomach and duodenum. Hence, pathways interfering with PG synthesis will likely cause gastric ulcers too, along with the desired objective of analgesia or antipyresis.

Another fine example of ion trapping is that of the anti ulcer drug omeprazole. The acidity of the stomach is due to the secretion of hydrogen ions (H+) by the parietal cells (oxyntic cells) of the stomach. This secretion is helped by an energy-driven pump, called the proton pump (H+/K+ ATPase), since H+ ion is same as a proton. Drugs such as omeprazole, pantoprazole, rabeprazole, esomeprazole and other such drugs inhibit this pump; and hence grouped under proton pump inhibitors (PPI).

Omeprazole, for example is a prodrug (has to be converted in the body to be activated). It is alkaline in nature and has to be given in an enteric coated form so that it can not be degraded by the acidity of the stomach. It dissolves in the alkaline environment of the small intestine, and as expected, it does not ionize. By not ionizing, it behaves as lipophilic and gets absorbed across biological membranes; is transported by blood to be secreted in the parietal cells. There, in the apical canaliculus, where the pH is very low (acidic), it dissociates into the active sulfonamide cations and consequently gets trapped (cations are positively charged ions, so named because they are attracted towards the cathode), which then cause disulfide linkages within the H+/K+ ATPase molecular structure to occur. The proton pumps get blocked. Acidity of stomach decreases. Ulcer healing is facilitated. Physics, physiology, chemistry, medicine are all inter related. United we stand.

Last Modified: Mar10,2014

To add Spice: Don't "Bend it like Beckham"

In my previous article, On her majesty's service..., I wrote about how the sperms 'sniffed' and chased their way to the ovum till it nailed her down. But for the sperms to reach it's destination, it has to be deposited in the right place. Thats where this organ comes into functioning. Its name is penis. It is a very interesting organ. Just like the states of matter (solid, liquid, gas and plasma), it can be soft, rubbery, and small or it can be hard and long. It not only injects (ejaculates) sperms, but also it acts as a conduit for urine to flow out of our system. Hence it has been christened as the genitourinary organ.

When mosquitoes bite us, their proboscis get hard, for a soft one can not penetrate our skin. Likewise, it needs a hard (erect) penis to enter the vulva. You would say, its nothing great; the muscles there would do this job. Here, we have no skeletal muscles, only smooth muscles are there. When we are stimulated erotically, say by seeing an erogenic picture/video or in our vivid REM sleep dreams, the brain sends the corresponding electrical impulses through the nerves (nervi erigentes). These nerves secrete (neurotransmitter), neither nor-adrenaline, nor acetyl choline and hence are classified into a separate group called the NANC fibers. They secrete a chemical substance called nitric oxide (NO). (Nitrogen has five valence states and thus forms 5 different compounds with oxygen; nitrous oxide, N2O, is another compound that is used in medicine, as an anesthetic). This gas, previously called EDRF or the endothelium-derived relaxing factor, relaxes non-vascular smooth muscles in the corpora cavernosa of the penis, so that more blood can rush through it. The penis gets tough, as a result. Nitrous oxide stimulates/activates an enzyme called guanylyl cyclase to form cyclic GMP (guanosine mono phosphate). It is cGMP which brings about erection. Well, this (cGMP) is finally broken down by an enzyme, called phosphodiesterase and the erection ends. Sildenafil (the popular drug viagra), tadalafil etc inhibit phosphodiesterase, making the staff stay erected longer. It can act only if the nerve supply is intact. It also causes visual disturbances (impaired blue-green discrimination) as phosphodiesterase also plays a role in vision. Viagra can add vigor to the diabetics suffering erectile dysfunction. Bear in mind, erection for too long a duration can be hazardous, a condition known as priaprism. The penis may slough out! Viagra, can also be used to treat spasmodic arterial diseases such as Raynauds phenomenon (blanching, bluish and cold fingers;- in a nutshell), refractory pulmonary hypertension (raised blood pressure within the lungs resulting from vasospasm) and other diseases. However, since it dilates arterioles, there is a chance of hypotension, due to diminution in the resistance in the vascular bed, especially in persons taking concomitant drugs, such as nitroglycerin (used for the treatment of cardiac pain or angina) and antihypertensives (blood pressure lowering drugs). A few cases of myocardial infarction or heart attack have been reported. Prostaglandins such as alprostadil; phentolamine and apomorphine can help us add spice to our lives.

I would do injustice, if I did not mention how the testicles are kept in an air-conditioned chamber called scrotum, in this context. There is a heat radiator system that operate in the scrotum which keeps the temperature 2 degree Celsius (centigrade) lower than the core body temperature. It is effected by the position of the testicles (which is not in direct contact/apposed with the body) and the radiator fins just mentioned (the pampiniform venous plexus). This is necessary for effective spermatogenesis to occur. Persons who wear tight pants, works in hot environment (furnace workers, vehicle drivers exposed to engine heat) have impaired spermatogenesis and the chance of infertility (NOT impotence) is great in them.

Lastly, about the nomenclature of this post. 'Bend it like Beckham' is a movie about the legendary football player David Beckham and Victoria (of Spice Girls fame) is her wife. How Beckham makes the ball bend in 'mid air' can be explained by Magnus effect, which I have discussed in my previous post. I hope I have explained my stand.

INSULIN : Take the pen to beat the pain

One of the greatest discoveries of the twentieth century which led to their discoverers having the Nobel prize (and sharing it!), this molecule, insulin, is secreted by the beta cells of the islets of Langerhans in the pancreas. It is produced as pre- pro-insulin by genes located on the chromosome 11. It is cleaved and folded to give insulin and C peptide before its release into blood. The pancreatic beta cells have a glucose transporter, called GLUT2 in their cell membrane which senses the blood glucose level and acts accordingly. When blood glucose level goes high, as after meals, glucose enters the beta cells through GLUT2. Thereafter, it is converted to ATP within the cell by metabolism (TCA cycle). This ATP then chokes (inhibits) the ATP sensitive potassium channel located in the cell membrane. Since there is more potassium inside than on the outside, the channel when open allows potassium to go out of the cell. Thus, glucose entry would mean more ATP, and more ATP will close this channel resulting in potassium build up with in the cell. Potassium (K+) being a positively charged ion (cation) will thus decrease the electronegativity inside, causing the cell to fire. As a result, voltage gated calcium channels open. Ca+ plays instrumental role in the exocytosis of insulin (insulin now comes out of the cell into the bloodstream).

Once it has come out, it circulates in the bloodstream and reaches the target organs/tissues. There it binds with the insulin receptor. The receptor molecule consists of 2 alpha chains (extracellular) and 2 beta chains (partly located extracellularly, partly transmembrane and partly intracellularly). The chains are linked together by sulfur atoms. Insulin binds with the alpha chains (naturally, as they are on the outside) of the receptor molecule, which in turn changes its (receptor) conformation. This triggers tyrosine kinase enzyme activity within the cell. This enzyme is intrinsic to the cell, only its activity is triggered by insulin. Then this enzyme autophosphorylates the receptor (meaning that the receptor itself gets phosphorylated: sounds incestuous!), and molecules such as IRS (insulin receptor substrates) are formed. They in turn phosphorylate/ dephosphorylate cellular proteins to bring about their effects. For example, activation of PI3 kinase (phosphatidyl inositol 3 kinase) results in the translocation of GLUT4 from the cell interior to the cell membrane. This transporter is important in the transport of glucose to the interior of muscle and fat cells. Thus GLUT4 is very important for glucose to enter these cells. Apart from this, insulin also has mitogenic action (by virtue of MAPK activity).

Thus absence of insulin will lead the cells to use fuels other than glucose. Thus the body is depleted of its fat stores, ketone bodies : a byproduct of fat burning are formed, muscles become wasted, and various complications occur.

Previously, insulin from animals (beef, porcine) were used to treat diabetes. Nowadays human insulin from recombinant DNA technology are being increasingly used. They are almost always injected subcutaneously, that too by a device called insulin pen (whence the name of this article). A pen has several disposable needles and a cartridge which may or may not be replaceable. Other routes of insulin administration has been explored, such as nasal, gastric, intrapulmonary by aerosol. Of these, the last mode of delivery holds promise. Insulin has been classified into short acting, intermediate acting and long acting insulins, according to the duration of action, of the type of insulin in question.

Therapy of diabetes often requires more than one component (type) of it (insulin), and is often based on SMBG and HbA1c and clinical parameters, rather than simple calculation on body weight or surface area basis. One example of how different types of insulins are mixed and administered has been sited in my former article, water water everywhere... This is just one example (actual treatment will depend upon SMBG, HbA1c and other informations, as cited above and more).