Monday's Molecule #16 is 1,1,1-trichloro-2,2 bis(p-chlorophenyl) ethane, better known as DDT. DDT is a powerful insecticide. It binds to the voltage-gated sodium channel and locks it in the open state. Prolonged influx of sodium ions causes the nerves to fire repeatedly and this causes death of the insect.The reason DDT is so powerful is due to its specificity. It binds to insect channel proteins but not to those of other animals (or plants, fungi, protists, and bacteria). Thus, it is an effective insecticide used to fight malaria and other insect borne diseases.
Unfortunately, even though DDT is not immediatly toxic to other animals it does have one disadvantage: it is extremely stable—its biological half-life is about eight years. Furthermore, DDT is stored in fatty tissues and its buildup in birds and fish resulted in considerable loss of these species. That, coupled with the evolution of DDT resistant insects, led to a ban of DDT in most countries by the 1970's.
Rachel Carson is largely credited with launching the environmental movement in 1962 with the publication of Silent Spring. The title refers to a world without birds. While I was writing this up I did a quick survey of the graduate students in the nearby labs and none of them had ever heard of Rachel Carson. Not only that, neither had several of my colleagues. I feel old.One of the main targets of Silent Spring was DDT. By the time the book was published it was estimated that DDT had saved the lives of millions of people through prevention of malaria and thyphoid but it's effectiveness was much diminished. That's why the ban was not as controversial as it might have been.
Let's look at the biochemistry of DDT. We have already learned about the simple voltage-gated potassium channel. The Na+ (sodium) channel is closely related to the K+ channel protein. Recall that the K+ channel consists of four identical subunits surrounding a central hole through which K+ ions enter the cell.
The Na+ channel protein is much larger than the K+ channel subunit because it consists of four of the smaller subunits fused into a single polypeptide chain. The tolopology of the Na+ channel protein is shown below.
Each of the domains (I-IV) corresponds to a single subunit of the K+ channel. Like the K+ channel, the four domains of the Na+ channel protein are arranged around a central tunnel through which sodium ions enter the cell. The S5 and S6 helices line the tunnel.The toplogy diagram above shows the locations of mutations conferring resistance to DDT and similar drugs. Each one represents mutants identified in resistant houseflies, fruit flies, mosquitos, or moths. The important mutations are substitutions at the 932 position normally occupied by leucine (L932) and at the 929 position normally occupied by threonine (T929) (blue dots). For example, the substition of isoleucine for threonine at 929 (T929I) confers almost complete resistance to DDT.
Incidently, the methionine at 918 (M918) is what confers sensitivity to DDT in the insect voltage-gated Na+ channel. Other animlas have a different amino acid at this position and they are not sensitive to DDT.
O'Reilly et al. (2006) modeled the structure of the Na+ channel using the known structures of the K+ channel proteins. This is necessary because the Na+ channel has not been crystallized. It's an excellent way to get a structure when you know that two proteins are homologous (descended from a common ancestor).
From the model, the authors were able to focus on the probable site of DDT binding based on the known mutations to resistance. In this case, they looked at the interface between helix S5 in Domain II and nearby helices S6 from Domain II (IIS6) and S6 from Domain III (IIIS6), which packs against IIS5 in the structure. They tried docking various insecticides in this region and came up with a good fit in all cases. The DDT binding site is shown below.
Note that the side chains of T929 and L932 interact directly with DDT. These are the sites of mutations to high levels of resistance. It looks like changes to these amino acids prevent binding of DDT and that's the basis of resistance.The S4-S5 linker helix is shown in yellow in this figure. Recall that this is the helix that responds to membrane potential by reorienting to a more vertical position. This, in turn, shifts the S5 and S6 helices to more vertical postions and closes the channel. In the presence of DDT the S5 and S6 helices are effectively cross-linked and they cannot shift to a position where they move closer together. This prevents closing of the channel. DDT locks the channel in the open conformation leading to a continual influx of Na+, uncontrolled firing of the nerve, and eventual death.
O'Reilly, Andrias O., Khambay, Bhupinder P. S., Williamson, Martin S., Field, Linda M., Wallace, B. A., and Davies, T. G. Emyr (2006) Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem. J. (2006) 396:255–263.
OK, I'm not quite as old as you are, but I've heard of Carson -- as a child, I was given The Sea Around Us and The Edge of the Sea, both of which I loved, and re-read several times.
ReplyDeleteBTW: I also not only remember the Joni Mitchell song (incl the DDT -- and do you know what the "big yellow taxi" refers to?), I can play it on guitar and sing it (albeit about two octaves lower ;-).
No, I don't know what "big yellow taxi" refers to. In fact, until yesterday I didn't even know that was the name of the song.
ReplyDeleteIt might be an urban legend, but it has always (or at least since 1970 when the song came out) been my understanding that the "Big Yellow Taxi" referred to the yellow cars that the Toronto Police (or as they were called then "Metro Police") used in the 1960s and 1970s. Certainly it is much more consistent with the sinister happenings in the rest of the song to have one's old man taken by the police instead of just going off for a taxi ride.
ReplyDeleteAccording to Wikipedia:
ReplyDelete"In the song's final verse, the political gives way to the personal, as Mitchell recounts the departure of a lover in the "big yellow taxi" of the title."
Interesting. So from the looks of things this wouldn't block the "ball and chain" inactivation of the channel. The Na channels will still inactivate and the cells will return to their resting state, but will fire again immediately after enough of the cells have deinactivated. That way you can get repeated action potentials instead of just have the channels constantly open and draining the potential gradient immediately.
ReplyDeleteSpeaking of agents that block the voltage-gated sodium channel, a Japanese restaurant around here has started serving fugu. Even though they promise the chef will sample it first- no thanks.
ReplyDeleteHumph - another lovely theory torpedoed by incovenient facts (assuming Wikipedia's veracity).
ReplyDelete"Rachel Carson is largely credited with launching the environmental movement in 1962 with the publication of Silent Spring."
ReplyDeleteOT, but for some reason a single media event can sometimes trigger a cultural movement. One of the earliest such events in Sweden must be the publication of "Lort Sverige" (roughly 'Dirty Sweden').
The continuing poverty of parts of early 20th century Sweden put its mark in dirty living conditions, poisoned local environments and subsequent sicknesses. (Remember, this is the Sweden that a century before provided US immigrants, that previously didn't own land or land good enough to even give consistently enough potatoes to avoid famine or poverty, much like Ireland.)
The book is said to describe all that. AFAIK it was responsible for triggering the push for cleaner and healthier living conditions and environment in the nation.
Gah! Thinking with my english brain here. Of course it was "Lort-sverige". But it also turns out it was really a series of radio shows in 1938.
ReplyDeleteGreat post. There’s another interesting, quirky and revealing fact about this system. In vertebrates, the sodium channel alpha subunit (the channel shown by Larry) is associated with smaller auxiliary so-called beta subunits. These beta subunits fine-tune the rate of opening and closing of the channel and enhance the expression of the channel at the surface of the cell.The sequence of these vertebate beta subunits reveals homology to a family of proteins called ‘cell adhesion molecules’.
ReplyDeleteThe insect sodium channel also has an associated small auxiliary subunit (tipE) that has a similar function to the vertebrate betas. However, the insect ‘beta subunit’ has no obvious sequence similarity to its vertebrate counterpart.
This seems likely to be another example of convergent evolution at the molecular level. In separate animal lineages, evolution independently used two different solutions to solve a similar need.
This makes no sense at all from an ID perspective – unless that is you assume the Intelligent Designer built the sodium channel in a fit of absent-mindedness!
Tony Jackson says,
ReplyDeleteGreat post. There’s another interesting, quirky and revealing fact about this system. In vertebrates, the sodium channel alpha subunit (the channel shown by Larry) is associated with smaller auxiliary so-called beta subunits. These beta subunits fine-tune the rate of opening and closing of the channel and enhance the expression of the channel at the surface of the cell.The sequence of these vertebate beta subunits reveals homology to a family of proteins called ‘cell adhesion molecules’.
It's interesting that the β subunits of the potassium channel are on the inside surface and they are NAD(P)-dependent oxidoreductases.
The β subunits of the sodium channels, on the other hand, are external membrane proteins that associate with the external part of the sodium channel. It looks like the β subunits fold via a disulphide bridge into a compact structure.
This single domain stucture in the β subunit may resemble one of the domains in a number of cell surface proteins, notably cell adhesion molecules.
There is some weak amino acid sequence similarity between the β subunit of the sodium channel and some of the domain regions in these adhesion molecules but the similarity does not rise to the level where we can conclude that the proteins (genes) are homologous (Isom et al. 1995).
Isom et al. (1995) suggest that the β subunit may play a role in localizing the sodium channels to the nodes of Ranvier during myelination. This makes sense to me. Tony, do you have any information on this possibility?
Hi Larry. The vertebrate sodium channel betas all have an N-terminal extracellular domain, a single transmembrane domain and a short intracellular C terminal sequence. It’s the N terminal extracellular bit that shows weak sequence similarity to an immunoglobulin domain (includes cell adhesion molecules like Thy 1 etc).
ReplyDeleteThe reason we think they are probably all distant homologues (members of the immunoglobulin superfamily if you like), is that key structural features like the crucial disulphide bridge characteristic of all Ig domains is there in the betas at just the right location (in fact a muutation that abolishes this bridge in beta 1 causes a form of epilepsy). Also, other structural features characteristic of Ig domains are present in the beta sequence exactly as we expect to find them. We can also use the known structure of the Ig domain to model the beta quite well too.
Um…see Morgan, K et al., PNAS. (2000) 97, 2308-13. :-)
“Isom et al. (1995) suggest that the β subunit may play a role in localizing the sodium channels to the nodes of Ranvier during myelination. This makes sense to me. Tony, do you have any information on this possibility?”
Yes, I think that’s right. As it happens, I’m putting together a review for Traffic about this very topic. I’ll send you a early draft if you like..
Tony says,
ReplyDeleteThe reason we think they are probably all distant homologues (members of the immunoglobulin superfamily if you like), is that key structural features like the crucial disulphide bridge characteristic of all Ig domains is there in the betas at just the right location (in fact a muutation that abolishes this bridge in beta 1 causes a form of epilepsy).
This is one of my pet peeves. As far as I'm concerned, structural simlarity does not trump sequence similarity when it comes to making decisions about homology (common ancestor).
If the sequence similarity isn't sufficient then it's best not to conclude that similar structures are "conserved" from a common ancestor. That conclusion implies the corollary that, for some reason, the structure has been preserved while amino acids were substituted at an extermely fast rate in order to abolish all traces of significant sequence similarity. That doesn't make much sense if you think about it—especially when you're dealing with proteins/genes that have only arisen recently.
It's just as reasonable, in my opinion, to conclude that the structural similarity is due to convergence. Since we can't decide between the two alternatives, we should avoid words like "homology" and "conserved" because they assume a conclusion.
Yes, I think that’s right. As it happens, I’m putting together a review for Traffic about this very topic. I’ll send you a early draft if you like.
That would be cool. I'll put it in the next version of the biochemistry textbook.
“This is one of my pet peeves.”
ReplyDeleteUm, yes … I see now that it is. And actually I understand where you’re coming from and I do agree with you in principle. But in the specific case of sodium channel betas, there are independent lines of evidence that suggest convergence with other members of the Ig superfamily is somewhat less likely than common ancestry.
So for example, it’s worth stressing that there are key sequence features always found in Ig domains and that we know have important roles in the structure, and that are also all there in the betas and in the same places compared to other Ig molecules – it most certainly isn’t just the disulphide bridge.
When we look at the exon structure of the beta genes, there are also clear boundary identities with those for some other Ig domain proteins (eg myelin P0), and that’s asking a lot for convergence.
Moreover, there’s biochemical evidence that at least one of the sodium channel betas is also a cell adhesion molecule in its own right, independent of its role as a sodium channel auxiliarly subunit, and the interaction takes place via its Ig domain exactly as expected for this structure.
By contrast, one of the key features of convergent evolution is that there are many different ways in which a particular functional structure can arise, and it’s these quirky differences in detail that gives the game away. That was the point of my original post (which kind of got sidelined a bit here). In insect sodium channels, their ‘beta’ is completely different from the vertebrate equivalent – the fly ‘beta’ sequence for example predicts a protein with two transmembrane domains, small N and C terminal intracellular sequences and an extracellular sequence completely unrelated to vertebrate betas or Ig domains. That really is strong evidence for convergence – and that’s interesting.