Saturday, June 2, 2012

A pore at the core of nerve action

A voltage-regulated ion channel protein studied in all its quivering atomic detail.

One of my earliest interests in biology was the nature of membrane proteins- how they could conduct ions back and forth across the membrane, thereby giving us energy as well as mental activity. One of the most obscure mysteries in the field was the dauntingly-named voltage-gated channel.

Nerves don't conduct electricity the way wires do. They are messy and biological, after all. Their conduction is far slower, but at the same time continuously refreshed as the signal moves along. The mechanism starts with a nerve membrane that is poised with an excess of sodium (Na+) on the outside, and an excess of potassium (K+) on the inside. This is the product of a pump (the Na/K exchange pump) that is always working away, sort of like the sump pump of the nerve cell.

The ion distribution is such that the resting state of nerve cells as well as most other cells is slightly charged, at -70 mVolts. If you open up the membrane to K+ ions only, this potential moves to -90 mV, and if you open the membrane to Na+ ions only, it moves to +100 mV. So the Na+ imbalance is significantly higher than the K+ imbalance.

The magic happens when a faint electical impulse comes along from upstream, and is felt by the special proteins mentioned above- channels (proteins that let ions through passively) that are gated by voltage- i.e. turned on when they feel a slight tingle. First, Na+ voltage-gated channels open, and dramatically let Na+ into the cell, which reverses the membrane polarity (the nerve fires, or spikes) to +40 mV. Then they close, and a different protein- the voltage-gated K+ channel- opens after feeling this positive charge. This restores local order, bringing the voltage back down to -70 mV. Indeed it brings the voltage slightly beyond, (called hyperpolarization), which prevents nerve impulses from going backwards, forming the so-called refractory period.

Action potential, in gory detail. The signal is travelling to the left, and some of the gating (Na+ and K+) on different protein channels is shown in seqeuence. The Na+ gates open when their "gating threshold" is reached, here at -55 mV. They close when the voltage rises further to +30 mV, when the K+ gates open.

A recent paper described in atomic detail how the K+ channels work, responding to a voltage change across the membrane they sit in, by shifting their shape in a complex way that alternately opens or closes their central pore, which is itself selective for K+ ions. How a protein channel can even be selective for K+ vs Na+ ions with their identical charge and similar shape is, incidentally, another highly interesting tale of protein structure & chemistry.

Author's cartoon model of the K+ channel's open state, with the critical S4 helix popped up and the channel opened, seen from the side in cut-away. K+ ions are in green, and the four channel subunits are shown on the right from a top view, with the voltage-sensitive modules in blue/red and the central pore in tan.
Author's cartoon model of the K+ channel's closed state. The S4 helix has descended and the regulatory domains have moved slightly away from the pore.

The key to this protein's dynamics is a part of its structure (helix S4, shown above as a purple rod) that has numerous positive charges on arginine and lysine amino acids along its length. This helix has been studied for some time, and its charges have been called the "gating charge", or "voltage sensor", following electrophysiological studies in bare membranes that showed that this segment of the protein, even in the absence of ions, accounts for some charge movement when one imposes an electic field, presumably being some important part of the protein that is itself charged and moves physically in response to an imposed voltage. A crystal structure has also been out for a few years, showing the static structure of this protein in open conformation.

The new paper is a tour-de-force of computation, taking these structural studies to the next dynamic level through atomic-level simulation of key parts of the protein over times up to 230 microseconds. Molecular dynamics is a well-developed field, though rarely deployed to protein-size chemicals, since the computational demands are so high. It resembles other kinds of compulational simulation, like aereodynamic airplane part simulation, weather simulation, climate simulation, etc., though it is very much on the more rigorous end of the spectrum, since basically all the relevant variables are understood.

In essence, it models each atom with its various chemical bonding, quantum mechanical, van der Waals, hydrophibic, and electrical characteristics, plus the jiggling of Brownian motion, to calculate by brute force a known chemical structure evolving through time. The method is commonly used by drug companies to "dock" drug candidates with their protein targets to see whether they might be effective lock-and-key matches. for instance.

In this case, the researchers started with known structures of the open channel, and imposed a simulated electrical field as would occur across the membrane the channel resides in to close it. They offer several movies of their simulations, two of which I link below, and believe have open access.

Starting frame from simulation, with open pore.
Ending frame from simulation, with closed pore, and red helices jutted down.

The pore of the channel is in blue at center, and begins in the open configuration. The voltage sensing domains are symmetrically arranged outside, linked by the critical red helix S4, which is the main voltage sensor. If you advance the movie rapidly by hand, you can note more easily the transition from open to closed. At the movie's native rate, the stochasitic Brownian motion overwhelms the closing movement, but is certainly interesting for that sake to show how proteins work in detail.

Another simulation frame, closeup of the sensor helix S4 from the side, in popped-up condition.

Above is a side view, of the voltage sensing S4 helix and its domain, but not the channel it regulates. The linked movie is a composite of two simulations, one from open to closed, then the reverse, from closed back to open. The colored bar at the bottom of the movie shows the charge being applied, which switches halfway through from red (more negative inside; hyperpolarization) to blue (more positive inside; depolarization). The rate is not uniform in the video, but the time elapsed is marked. Note how the critical arginine side chains (positively charged, with blue branched ends) work their way stepwise down the stable green helix by interacting with negatively charged partners.

As the researchers state, using the abbreviations R for amino acid arginine and K for lysine, both positively charged: "The S4 helix-bearing gating charge residues R1, R2, R3, R4 and K5- is the main VSD [Voltage Sensitive Domain]  moving part. S4 translated ~15Å overall across the membrane in sequential steps while rotating ~120°, moving in a groove formed by the largely stationary S1 ro S3a helices."

The S4 helix links to the pore in a couple of ways, first through its end, joining to a linker colored above in yellow, which tugs the inside end of the pore. Secondly, the four protein blobs that hold the S4 helices rotate around a bit in response to the S4 helix movement (evident in the first video sequence), in a way that helps the rest of the pore either open or close, in a sort of origami dynamic.

There is much more going on here than can be briefly related, but this gives you a taste for the main points, the explanatory power, and indeed the beauty of such a detailed analysis of an important biological structure.

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  • Economics image of the week- we continue falling behind where we could be:

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