Superhero aquaporin

How does water get into and out of our cells?

Water is an amazing molecule. Common, but complicated. Clear, essential, and taken for granted, but also chemically very weird. Water is more prominent as one goes out in the solar system. The Oort cloud is a vast, cold swirl of ice and dust, from which Earth is thought to have obtained most of its water by bombardment, after the original light gasses were driven off in the earliest hot phase of planet formation.

We are 60% water. Biology originated in water, and all biochemical reactions take place in water as the solvent. One of the first steps to the origin of life may have been controlling water by forming oily membranes to separate inside from outside, creating primordial cells.

Biological membranes are very slightly permeable to water, and less so as one adds cholesterol. By the time one gets to all existing cells in the evolutionary story, there is a need for special proteins that conduct water through the cell membrane at a high rate. These are all passive pores, (not pumps, which would require extra energy), though they can be regulated in various ways to turn on and off. Our kidneys are full of them, with at least six different genes encoding water pores, called aquaporins.

Cells typically regulate their water balance not by pumping water itself around, but by manipulating ion concentrations (of sodium, potassium, and chloride, principally) within. They can build internal water pressure (turgor pressure) by pumping an excess of an ion inward, and waiting for diffusion to attempt to equalize the concentrations inside and outside by sending water after the ions into the cell.

But here is where the aquaporins come in. In bare membranes, this diffusion process is way too slow for most cases of regulation, so cells need a passive, specific, and high-volume way to let water in and out, faster than the membranes themselves allow. And the toughest part of this is making such a channel specific to water alone. Making a hole in a membrane is not difficult. Many pathogens do just that to kill cells. An aquaporin needs to conduct H2O only, not salts, not hydrogen ions (protons), and not OH- ions, either. It is a job for a very intricately structured protein.

A recent paper described a very high-resolution X-ray structure of an aquaporin from yeast cells, taken as a model for all kinds of aquaporins. Typical protein structures come in at around 2Å resolution, which is enough to follow a protein backbone around and get a good idea of the overall protein structure. This one is 0.88Å, a remarkable achievement for membrane proteins, which tend to be very difficult to crystalize and solve (prior work). 

The quality of this structure is so high that the researchers can tell the difference between an electron cloud shared (conjugated) between the carbon on amino acid Gln137 and its oxygen (red), and an electron cloud more evenly shared elsewhere in the structure, between the carbon on Glu51 and its two oxygens. X-ray crystallography provides only electron densities, from which the identities of the associated atoms and molecules must be deduced.

The overall structure determined by this group, in cut-away cartoon view, with water molecules as red dots. SF is the "selectivity filter", and NPA is another well-studied area, with a "asparagine-proline-alanine" protein sequence.

This resolution allowed the researchers to see individual water molecules snaking their way through the central channel (at least very roughly, there was still some motion that blured them out) in the low-temperature frozen crystals, something very rare in protein structural studies. Because yes, as you would guess, aquaporin channel is a big protein structure that exists entirely to create a narrow channel running through its center.

Youtube provides a computer simulation run by other researchers using another model aquaporin. Brownian motion of the peripheral waters dominates the video, while the waters inside the channel bounce around more slowly. But quickly enough to pass at a rate of billions per second! The channel shows distinct charged areas, blue (+) and red (-), which organize the slightly dipolar water molecules as they slip through. One of the water molecules is highlighted in yellow so you can track it. The simulation shows two particularly narrow regions, upper, near the large blue blob, and also a lower one, where the waters have to squeeze through.

"A single human aquaporin-1 channel facilitates water transport at a rate of roughly 3 billion water molecules per second."

Fine.. but anything could run through here, at least anything smaller than water, which being a three-atom molecule, means that most single-atom ions would be smaller. What makes this channel selective for only water? Here we turn from structure to electrostatics. The channel works by funnelling each water molecule through a channel that is not only narrow, but also lined in very specfic ways to test that each molecule passing is actually water.

Example of a site where one water (#4) is extensively coordinated to tell by shape and charge that it really is water.

"MD [computer simulation / molecular dynamics] snapshots illustrate how this geometry achieves exceptional water selectivity, because all four H-bond donor and acceptor interactions are filled as water moves through the SF [selectivity filter] (C). The presence of four closely spaced water-selective sites optimizes the aquaporin SF’s ability to discriminate water from other small molecules. Hydroxide ions, in particular, suffer a geometric penalty, because they cannot simultaneously donate H bonds to the backbone hydroxyl of Ala221 and to Nε of His212. Conversely, all H-bond interactions are distorted from ideal water geometry, and this avoids binding water too tightly, such that efficient transport is compromised."

A larger view of one of these "selectivity filters" is shown below, where side chains from the protein (in green and blue) reach out to touch the water molecules, shown smaller than their real effective size so that we can see what is going on. Pairs of waters are shown in progressive positions in the channel. The researchers conclude that the pair move in lock-step from one pair of positions to the next pair, all of which show up in the structure, but only two of which can be occupied at a time.

Pas de deux of two waters through the selectivity filter, which coordinates them closely with slight positive charges from Arg227 touching the negative water dipole on the oxygen, while the slight negative charges on Gly220 and Ala221 touch the positive dipoles of the hydrogens.

Which brings up the most sensitive issue, which is that water needs to be sensed very carefully on all sides to bring the right shape through, and kept from bringing along H+ protons, or coming through in OH- form- but not be held so tightly that it can't get through at all. So all these touches are done like in a car wash, on the fly as the water molecules come through one by one.

It is great to see basic scientists digging deeper into the basic mechanisms of life, unearthing knowledge whose application may be very far off, but whose beauty and elegance is a permanent and worthy achievement in itself.

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Injustice, hoodies, and gun nuts

Reaction to the Zimmerman trial.

Apologies for a political post, but I was surprised by the Zimmerman verdict. While second degree murder was indeed a stretch, a verdict of voluntary manslaughter seemed a foregone conclusion. When one participant in an altercation is dead, the remaining evidence is unavoidably tainted and partial. There should be a presumption of responsibility on the killer, not simply an assumption of innocence.

In this case, there was no question who killed whom, and who was armed. The only question was why, which led to the legal hairs being split down the degrees of murder. Zimmerman gave us very little to go on in the core of this case, not even testifying in his own defense. After doing everything he could to provoke a confrontation, he evidently got into a confrontation, and bit off a little more than he could chew, with Martin, (as the trial seemed to establish), beating him from above.

Those circumstances don't justify murder, as far as I understand it. If you start a fight, you don't get a free pass to murder just because the other person fights back. The whole "stand your ground" concept thankfully didn't come up in this case, because Zimmerman wasn't on his own ground, except in the guise of a neighborhood vigilante and faux-cop. So the defense was simple self-defense, based on a decided lack of evidence, the main source of evidence having been conveniently killed by the defendant, while the defendant was tellingly silent on the key events that precipitated the fight. It is a case where lack of evidence speaks volumes.

In the larger picture, we rely on our justice system to deter communally destructive behavior. And the kind of race-tinged vigilate-ism that this verdict approves is certainly toxic and destructive. America used to stand for law, justice, and, you know.. the American way. The South keeps getting in the way of such decent ideals, however. And justice in Florida seems to retain a distinctively Southern flavor, exemplified by this jury. A juror has said that Zimmerman's heart was in the right place, and that the jury was not racist at all. If only the dead could speak, eh?

Incidentally, this case is also a classic example showing that guns kill people. If Zimmerman didn't have a gun, both these people would still be alive today. The gun enabled an idiot to execute the target of his suspicions and hatred with little thought, or, more to the point, careful, polite, interaction. His emotions were to some degree understandable, in a dumbly profiling, stereotyping kind of way, since the neighborhood had seen several crimes over the prior few years. Still, the gun spelled the difference between amateur policing and murder.

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"But he warned that Congress itself remains the greatest obstacle to faster growth. Federal spending cuts are reducing growth this year by about 1.5 percentage points, he said. While the Fed expects the impact to diminish next year, he said there was a risk Congress would create new problems for the economy."

The women are at fault

Bernard  Lewis offers an hypothesis about Muslim cultural development.

Just before 9/11, Bernard Lewis published a provocative book about the Islamic world titled "What went wrong? The clash between Islam and modernity in the Middle East." There is very little discussion about the causes, but a great deal of historical detail showing that the muslim world has indeed fallen behind the West, (or North, or however one wants to term it), and is bitter and has long been perplexed about the situation.

To recap a bit from his conclusion:

In the course of the twentieth century, it became abundantly clear in the Middle East and indeed all over the lands of Islam thta things had indeed gone badly wrong. Compared with its millennial rival, Christendom, the world of Islam had become poor, weak, and ignorant. In the course of the nineteenth and twentieth centuries, the primacy and therefore the cominance of the West was clear for all to see, invading the Muslim in every aspect of his public and-more painfully- even his private life. 

Modernizers- by reform or revolution- concentrated their efforts on three main areas: military, economic, and political. the results achieved were, to say the least, disappointing. The quest for victory by updated armies brought a serious of humiliating defeats. The quest for prosperity through development brought, in some countries, impoverished and corrupt economies in recurring need of external aid, in others an unhealthy dependence on a single resource- fossil fuels. And even these were discovered, extracted, and put to use by Western ingenuity and industry, and doomed, sooner or later, to be exhausted or superdeded ... Worst of all is the political result: the long quest for freedome has left a string of shabby tyrannies, ranging from traditional autocracies to new-style dictatorships,  modern only in their apparatus of repression and indoctrination.

He makes the additional point that even while the Muslim world has been so bitterly conscious of falling behind, other countries, especially in Asia, such as Korea and China, have zoomed past them into modernity. What is the problem?

Lewis offers only the most off-hand comments to this question in his conclusion, but one of them caught my attention in the most riveting way:

"For others, the main culprit is Muslim sexism, and the relegation of women to an inferior position in society, thus depriving the Islamic world of the talents and energies of half its people, and entrusting the crucial early years of the upbringing of the other half to illiterate and downtrodden mothers."

This knits up so many threads that one's head spins. For instance, one issue is that Islam was once the most advanced culture, at least in the hemisphere, if not the world- open to Greek and Roman learning, building on it, and passing it on to others. For its time, Islam gave high regard to women, with rights to inherit and have their own property. Even polygamy was not originally formulated as an excercise in male dominance and competition, but of caring for widows and other isolated women (of whom there were many after the wars of Muhammed) who might otherwise become beggars or prostitutes.

So one might say that Islam was at one time in a leading position with regard to women's rights, and as long as that held, it also held relative cultural leadership in the broadest sense. One can well imagine the influence that educated and civically engaged women have on their children, and the converse effect that a relentless confinement to family, clan, and tradition have. As previously noted, our parents give us meaning, and they also provide us with horizons and ambitions, whether small or large minded.

Another issue is the relative development of different countries, especially within the Islamic world. Why is Afghanistan saddled with the Taliban, while Egypt has the more moderate, though still a bit crazy, Muslim Brotherhood? Could the relative oppressiveness of the burkha and the veil (and all that they signify about the position of women) have something to do with it?

The fight for women's rights in the Islamic world is not just a matter of goo-goo feelings and Western domination, but addresses the very core of cultural development in the long term. One can hypothesize that every gain that women make in education, cultural engagement, and rights, yields, a generation down the road, a society less prone to blood feuds and more engaged in further education and higher cultural development, built by men and women inculcated with the ideals of their mothers.

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Our plastic memory process

Dynamic and adjustable memory associativity in the brain.

A funny thing about memory is that it bleeds. It takes on schematic properties that allows other concepts to associate with it, notoriously as free association, but also during learning, trauma, metaphor, and creativity. PTSD is a problem where too many things trigger bad memories and those bad memories generalize to mean far more than the bare incidents in question. If the memory of one distasteful event is to be useful, its core elements need to be recognized and generalized to enable avoidance not just in that exact setting again, but of future patterns that are similar to some regulated degree. So how similar is similar?

Surprisingly, memory generalization can be studied, and a recent paper described connections between the hippocampus, frontal cortex, and an intermediary structure called the nucleus reuniens, showing that some of these connections affect the degree of memory generalization when either destroyed or enhanced. The nucleus reuniens is part of the thalamus which is smack in the middle of the brain, with important roles in consciousness and alertness, among much else.

This is all done with mice, using cutting edge techniques. The test was to have the mice experience a shock in combination with an alert sound in one cage, and then to test response to the same sound (tone condition) or to no sound in various settings, like the same cage, or similar cages, or a somewhere completely different. Normal mice do not show fear outside the original cage context, unless you ring the same tone that was  associated, Pavlov-style, with the original fear training. But mice that have had their medial prefrontal cortex areas ablated (mPFC), or their nucleus reuniens ablated (N. reuniens), show continued fear in new cages. I could not tell how universal this fear expression was ... were the mice just totally frozen now, fearful of all conditions? That was not clear.

Genetic ablation of various mouse brain structures specifically affects the degree of fear memory generalization, as tested by fear expressed in an altered context, relative to the original context (cage) or the original tone sounded during fear training. The role of the medial prefrontal cortex (PFC) had been known previously, but the equally important role of the nucleus reunions had been unknown.

At any rate, the coup de grace was for the researchers to use some clever molecular technology to ablate only the neurons connecting the PFC with the N. reuniens, by injecting the mice with one genetic element of a lethal cocktail (a gene for tetrodotoxin hooked up backwards to a promoter of expression) in the mPFC, and the other element (a recombinase that flips the toxin cassette into the expressing orientation). Each was labeled with a fluorescent protein to show where the injections took place:

Locations of injection of two genetic constructs, in a mouse brain section, whose products combine to destroy connected neurons. Green shows the medial prefrontal cortex, injected with the flipped toxin gene, and pink shows the N. reunions, injected with the recombinase gene that activates toxin expression.

Neurons transport this kind of expressed protein through their cell bodies, even out to the farthest axons. They also let some into the synaptic vesicles that send neurotransmitters across the gap between neighboring neurons (axons and dedrites). This means that a small amount of the recombinase would be transported from the injected region into the target area where its projections go, and be able to induce expression of the toxin in those target neurons that received the complementary injection, killing them. The figure shows the result, where control mice with non-expressing injections show normal fear-context dependence, while mice that got the full treatment in the nucleus reuniens and prefrontal cortex show more generalized fear, like mice that had their whole nucleus reuniens ablated.

Specific ablation of neurons connecting the mPFC and N. reuniens results in expanded memory generalization (TetTox bars, green), while neuronal activity enhancement (NL2KD bars, blue) in the same neurons generates the reverse effect of lower memory generalization. Response to the tone or the whole training context remains normal in all cases.

They even were able to do a converse experiment, using the recombinase to induce, instead of a toxin, a repressor of neuroligin 2 which represses neuronal transmission, essentially enhancing transmission between the two injected areas (the blue bars, above). These show the (modest) opposite response of lower fear response in the altered context (than control mice with sham treatment), with similar response in both the original cage and the trained tone. This provides quite a strong argument for the specificity of what they are seeing- that the nucleus reuniens is a critical way-point for signals from the prefrontal cortex that tell the hippocampus that a memory is relevant to more specific conditions than it might be inclined to apply them to otherwise. As in most things, the prefrontal cortex refines and inhibits our deeper brain processes.

Lastly, they used the very latest technology to convert the nucleus reuniens neurons to be light-inducible in their firing (optogentics), by injecting a gene for channel rhodopsin, which converts light into membrane potential which in the neuron can induce action potentials. This method allows researchers to run the affected cells at any firing rate they wish, up to 50 times per second, driven by a light guide directed right into the structure in the brain they are interested in.

Optogenetic experiment, where light is conducted into the site of an injected channel/rhodopsin gene which allows researchers to use strobed light to fire neurons at will, and also fluoresces red.

They used two driving methods, one a constant 4Hz (4 firings per second; tonic) and the other bunched in fifteen firings within a half-second, at five second intervals (phasic). The experimenters evidently came up with these patterns through trial and error, and they had opposite effects, when run while training the mouse to the fear condition (shocks to the feet in a cage). Running the tonic pattern during training yielded lower fear later in altered contexts (lower generalization), while the phasic pattern yielded higher fear. Neither one affected the response of the mice to the true fear condition of the original training context and/or tone.

Optic driving of the N. reuniens during memory training yields opposite effects on ultimate memory generalization, depending on the pattern of neuronal firing driven- phasic or tonic.

The idea from this work is that while the hippocampus stores specific memories, other connected areas allow those memories to be generalized to *similar situations, accounting for allegory and much else about our mental operations. There is a two-way circuit from the hippocampus to the nucleus reuniens and on to the medial prefrontal cortex, where memories exist in more abstract form. Here, the researchers show that for at least one type of memory, the obscure nucleus reuniens links the two, with an active role in whether a mouse's memories flood in during distantly related situations, or only in the more restricted context of the original experience.

Related mechanisms are likely to be relevant to the binding problem of how separate features of an experience or scene are linked by our minds into a unitary experience with various possible abstractions and composite or derived properties, i.e. consciousness. The way we associate new experiences with prior memories and knowledge, either freely or sparingly, should work similarly and be intimately connected with the degree and quality of our creativity.

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