Monthly Archives: October 2011

Ion Channels – a brief overview

I have been reading a book called “Principles of Neural Science” by Eric Kandel. I thought it would be a good idea to write a summary of a chapter that I read recently and tell you all about one the most important structures present in the cytoplasmic layer – Ion Channel. Ion channels allow the movement of ions across cell membranes, and therefore fundamental physiological processes such as muscle contraction.

Ion channels are membrane protein complexes. They are embedded in the lipid bilayer which is made up mostly of phospholipids, which have a hydrophilic head and two hydrophobic tails. The lipid bilayer is the barrier that keeps ions, proteins and other molecules where they are needed and prevents them from diffusing into areas where they should not be. They make good barriers because they are only a few nanometres thick, they are impermeable to most water-soluble (hydrophilic) molecules and are particularly impermeable to ions.

Definition: Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane by allowing the flow of ions down their electrochemical gradient.

Concept of Open/Close

Ion channels provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane. The channel, or pore structure, is said to catalyze the ‘reaction’ of transporting charged molecules across a low dielectric medium. The ‘catalytic site’, the central channel, is either open or closed. The conformational change between closed and open state is called gating. Channel gating is controlled by external factors like enzymes are controlled by modulators and effectors.

Types of Ion channels

There are over 300 types of ion channels in a living cell. Ion channels may be classified by the nature of their gating, the species of ions passing through those gates, the number of gates (pores) and localization of proteins.

  • Ligand gated channels
  • Voltage gated channels transmembrane potential
  • Second messenger gated channels
  • Mechanosensitive channels
  • Gap junctions

Structure of a channel

Channels differ with respect to the ion they let pass (for example, Na+, K+, Cl), the ways in which they may be regulated, the number of subunits of which they are composed and other aspects of structure. All ion channels are complexes of transmembrane proteins, sometimes they contain cytoplasmic subunits, often they are glycosylated. The 3-D structure of most ion channels is not known, with two notable exceptions, porins and a K-channel, both of bacterial origin. There exists, however, a multitude of biochemical and functional data, combined with mutagenesis experiments that give information about the transmembrane topology of these proteins, dividing it into transmembrane segments and extramembraneous loops/domains. Often size and location of loops on one side or the other of the membrane can be determined by chemically modifying the protein and analyzing which amino acids have been modified.

For example: Nicotinic Acetylcholine Receptor – nAChR

The structure of nicotinic acetylcholine receptor, has been determined to 0.9nm resolution by cryo-electron microscopy. The nAChR is a heteromeric glycoprotein complex composed of five integral membrane proteins in a stoichiometry of α2βγδ.

The five subunits are arranged in a circular fashion around a central hole that provides an ion pathway across the post-synaptic cell membrane. The pentameric complex has a fivefold pseudo-symmetry because its subunits are not identical. Acetylcholine binding induces the opening of the channel.

Fig: Pentameric arrangement of nAChR subunits

Problems associated with Ion channels

There are a number of chemicals and genetic disorders which disrupt normal functioning of ion channels and have disastrous consequences for the organism. Genetic disorders of ion channels and their modifiers are known as Channelopathies.

For example:

  1. Human hyperkalaemic periodic paralysis (HyperPP) is caused by a defect in voltage dependent sodium channels.
  2. Dendrotoxin is produced by mamba snakes, and blocks potassium channels.

That is it for now. I hope you liked what you read. If you are interested, I could always find more references and papers to support the above data.



Deadly Organism of the Fortnight: Pterois volitans.

This fortnight’s selection is an absolutely beautiful animal that happens to be extremely deadly, funnily enough.
It is a marine reef fish that is native to waters of the Indian and Pacific Oceans, but one that is also found in the Atlantic as an invasive species following accidental introduction.

Pterois volitans.

The species is commonly known as the Lionfish, and it is deadly because of spines that deliver venom. These spines are attached to glands and the whole bit is covered by an integumentary sheath that bears the same striking colouration that the rest of the body does. There are 13 dorsal spines, 3 anal spines & 2 pelvic spines.

Anatomy of dorsal venom-delivering spines in P.volitans

The illustration is taken from this paper, which unfortunately is behind a paywall, but if you can you may want to read the paper.

Now, if we were to focus on the venom per se for a little while, one would find that the venom is primarily cardiotoxic, i.e, it has both inotropic effects (weakening the force of contraction) and chronotropic effects (slowing down the heart rate) by virtue of acting on adrinergic and cholinergic neurons, and also by triggering the release of nitric oxide. You may find a reference that elaborates upon the exact mechanisms here.

cDNA analysis of the venom genes has been carried out, and it reveals that Pterois spp venom is very similar to stonefish venom, you can find more on that here

Talking of the venom, there was also a mention of a non-proteinaceous toxin in the literature, but given that toxin peptides have already been sequenced and studied using cDNA analysis, I’m not quite sure how important/correct that report was (See here for a reference)

They’re apparently popular aquarium fish. Yeah, right.

That is all from me insofar this article is concerned.

Isothermal Approaches to DNA Amplification.

I’ve already discussed PCR on this blog before. PCR is an adiabatic process that takes place at varying temperatures, which brings with it the requirement for accurate heating and cooling equipment to maintain and alter temperatures to predetermined set points, arguably, this makes precision PCR equipment extremely expensive.

Could there been an alternative method for amplification that could do away with complicated equipment?
The people who came up with HDA (Helicase Dependent Amplification) answered that question with an emphatic yes, and the process is isothermal.

So, how exactly does this method work?

As with PCR, HDA is also based on in-vitro DNA replication. Here, a primer, a polymerase and a helicase are employed.
The helicase unwinds the DNA template enzymatically, a direct consequence of which is that DNA needn’t be denatured by heating to a high temperature. The primers can then bind to the template and the polymerase in the reaction mixture can then bind extend the primers, and produce a duplicate copy. So you can put in one double stranded template, primers complementary to your target sequence at the extension start site, and a polymerase, and just set the temperature to the optimum level for polymerase activity. Ta-dah.

Schematic diagram of HDA. Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black triangle) separates the two complementary DNA strands, which are bound by SSB (grey circles). 2: Primers (lines with arrow heads) hybridize to the target region on the ssDNA template. 3: A DNA polymerase (squares with mosaic patterns) extends the primers hybridized on the template DNA. 4: Amplified products enter the next round of amplification. From reference cited below.

The system, when reported in 2004, was able to easily achieve a million-fold amplification. This is the relevant paper.

There you go, that is all from me on HDA. The paper has examples which show the method in action successfully amplifying template DNA, which makes it a potentially suitable method for the development of diagnostics.

There is also a related method called Recombinase Polymerase Amplification, which is also isothermal, and you may read more about it here.

The difference between HDA and RPA lies in the fact that the latter uses a recombinase, which is involved in exchanging segments. A brief visual summary of the method follows.



The fact that both processes can be carried out at 37’C means that they can be carried out in a bog-standard incubator instead of in one of those really expensive PCR thermocyclers (however, that might be beginning to change, too, with a $10 cycler that someone built in the USA).

That is all from me in this post. Happy reading.


Oncological complications – models surrounding tumour suppression.

Now that I’m formally studying cancer biology, there was always bound to be exposure to some rather fascinating things which I’d then go on to blog about, duly. Today, I will be looking at Tumour suppressors and how several things can take place with respect to how they are knocked out and how this can have varied implications for tumourigenesis.

When tumour suppressors were discovered, it was found that there were genes that were capable of causing cells to hyperproliferate if knocked out. This meant that there were genes in cells whose normal function was to prevent cells from growing too much or too rapidly. For tumour suppression to fail, it appeared that both copies of a gene had to be knocked out, which is why fusing normal cells with cancer cells almost inevitably prevented the properties of cancer cells from manifesting.

The first tumour suppressor that was identified was the protein pRb. This was identified by looking at people who got the childhood tumour retinoblastoma and subsequently finding out that certain families which showed an inherited predisposition to retinoblastoma had inherited a dysfunctional copy of the gene.

It was later found that if there was a viral infection such as an adenovirus, it would prevent Rb from acting by blocking its binding to a family of transcription factors called E2F. This evidence led to the identification of the complete loss of Rb function as one of the mechanisms for tumorigenesis, especially in retinoblastoma. Knudson, on the basis of this, proposed a two hit hypothesis; i.e, there had to be two events that resulted in the knocking down of both of the copies of Rb before tumour suppression was lost.

Explanation of Knudson's Two Hit Hypothesis.

The ways by which Rb function is lost is beyond the scope of the current post in any detail, but basically, unless phosphorylated by cyclin dependent kinases it binds to E2F, preventing it from activating the genes that are expressed to progress to the next phase of the cell cycle. If it is phosphorylated due to structural changes that prevent dephosphorylation, for instance, it loses the ability to bind to E2F in any case and therefore the cell cycle checkpoint it is active at fails.

How Rb works in regulating the G1-S checkpoint.

An early review of pRb function may be found here

Knudson’s original paper on the two-hit hypothesis can be found here.

Think the two hit hypothesis sorted questions on tumour suppressors out? Think again, later research showed that some tumour suppressors, such as the ever-so-important p53 and p17 for instance, acted in a gene-dosage dependent manner. A brilliant illustration of this was provided when people produced mutant mice that had one copy of p17 knocked out, and both copies knocked out. They observed that those with one copy knocked out demonstrated hyperproliferation that was intermediate between that of normal mice and that of those which had both copies knocked out. This was called haploinsufficiency, i.e, if you knocked out just one copy of a tumour suppressor, it could still have a significant phenotypic effect. If reading scientific literature is your thing, you can read more about the genetics of tumour suppressors and tumour suppression here.


So, could we then classify tumour suppressors into those that required two hits or those that worked by haploinsufficiency, with no overlap? Surely it made sense that losing one copy was not as bad as losing both copies?
As it turns out, not quite.

Emerging evidence has led to the formulation of a continuum model, which seeks to indicate that the type of event needed to knock-out tumour suppression can be dependent on other genetic circumstantial factors too. In the apposite paper, Knudson et al refer to PTEN, where haploinsufficiency is more tumorigenic compared to complete loss if p53 function is good because deletion of both copies will trigger a p53 mediated failsafe reaction that kills the cell. However, if p53 is knocked out, losing both copies of PTEN is a good idea for cancer cells.

Paradigms of tumour suppression. From Berger et al, Nature. (link in paragraph)

It would appear, as with most other things in oncology, context is key. That is all from me this time round.

Deadly Organism of the Fortnight: Phoneutria nigriventer.

This fortnight’s post has been a bit late in coming since I’ve been caught up in some academic work, nonetheless, here is a selection that will fuel the fears of arachnophobes. Phoneutria nigriventer is a species of Brazilian wandering spider, and while it is not as venomous as another species in the same genus, P.fera, I’ve chosen it for examination because it is well documented.

Phoneutria nigriventer; threat display.

As with the other candidates I’ve posted about in this series, it is venom that makes it deadly.

The venom’s main component is a peptide called PhTx3 , which is capable of blocking calcium channels that maintain the ion gradients needed by muscles to be able to contract properly following neural activity by motor neurons at the neuromuscular junction. This means that it can inhibit acetylcholine signalling and therefore trigger paralysis. If you wish to delve into the literature here, please see
this paper which details the evidence for the venom being a calcium channel blocker.

There is also a rather unsavoury aspect to P.nigriventer envenomation; it can induce priapism in humans, which is a condition where an erect reproductive appendage doesn’t return to a flaccid state for an abnormally long time; in this case, symptoms can apparently last for hours and may lead to impotence as an aftereffect.

This however also makes it a suitable research candidate for treatments of erectile dysfunction, go figure.
If you need help with that, here is the research paper that describes the confirmation of the activity of one of the components as a potentiator of erectile function in mice

If you’re interested in going through the amino acid sequences of the proteins in the venom, you may find the following database retrieval results sheet useful

So guess that is it for this fortnight’s deadly organism of the week.