The points which I had raised and you further commented on were not to say that charge-pairing does not occur. Rather that, although such a concept was proposed in the mid 1980s (or may be even earlier), even after 25yrs our understanding of such a basic proposition remains fraught with equal amounts of uncertainties!

Anyhow, the Pless et. al. paper – initially the paper actually does seem to be very exciting. But honestly after you’ve actually seen the supplementary materials as well, I just can’t help being left bewildered. Here’s why:
E293 neutralization with Nha: no effect GV. But kinetics of GV and FV are slowed down. Through some funny/unexplained calculations, the authors concluded that the pronounced deceleration of the kinetics (which by some weird way corresponded to a 3-fold change in the activation/deactivation kinetics) suggests its role as a “modest catalyst”. The way the paper is written it is worded in such a way so as to suggest that E to Nha substitution does not apart from change the charge, but E to Q makes a H-bond donor, a H-bond acceptor, thereby suggesting that solvation at the site plays an important role.
Dougherty’s group have clearly shown that the H-bonding capacity of Nha, relative to E, is ~2-3 kcal/mol weaker. If the effects observed on perturbation of the site are largely via solvation, then Nha would’ve had more of an effect (this is just speculation on my part).
The next point in this regard is that, E293 when mutated to Q produces steady-state GV, QV and FV curves which aren’t signficantly different from WT. The effect of E to Q mutation is large seen in the limiting slope analysis (Seoh et. al. Neuron 1996), where it was seen that this negative charge neutralization reduces Qmax from 13 to ~9. So my point is a similar experiment needs to be done with E293 to Nha to actually say whatever Pless et. al. want to say about this site.
Finally, about the role of this E293 in reducing Qmax – if you compare Aggarwal and Mackinnon’s values to Seoh et. al.’s values, there is a consistent discrepancy of 2-3 charge units for almost all the charge neutralizations. (The charge add back experiments by Ahern and Horn in JGP actually support Aggarwal and Mackinnon and are not in agreement with Seoh et al). Is the reduction of Qmax due to E293Q, real to begin with? Also it must be mentioned that the comparable site in the BK channels is a Tyr. Ma and Horrigan (JGP 2006) found that mutating this site to E does not have any effect on Qmax.
So, where are we about really understanding the role of E293 in the functional cycle of the VSDs – for me, actually nowhere!!!

Next for E283, Pless at. al.’s data and analysis is more palatable (but only slightly so). If you see the crystal structure of 2R9R, there are water molecules everywhere around the E283 site. How can charge pairing occur if there is water all around?

About the D316 site, I wouldn’t know what to make of it, if you consider the data in the supplementary information.

The other thing which I just want to mention that while stabilizing the charge is important. Keeping it a bit unstable is also as important, so that things can happen at the millisecond time scale. It is almost reminiscent of Dr. Mackinnon’s Nobel Lecture Video, where he talks about the paradox between flux of ions and the high selectivity of K ions. While the charges need some stabilization, I firmly believe that it is necessary to have forces attenuating such stabilizations as well, so that the energy barriers for the transitions are relatively smaller such that the charges can move up and down quickly as the voltage-changes.

It is clear that other channels don’t have the exact same pattern of charge compensation. I imagine that altering the distributions of charges within the membrane would be an excellent way for evolution to alter the gating behavior of the channel. Moving or removing counter charges in the membrane would affect the relative stabilities of the different channel states. More work certainly needs to be done to fully understand this potential mechanism. Also these different channels don’t all have the same number of gating charges so until we have more structures it is difficult to compare them directly.

Thank you for bringing up the very interesting work of Pless et al. I really should have included this paper in my discussion but somehow it slipped my mind. What they show is not that these residues have no role in stabilizing the different conformations of the VSD but that their role may not be mediated by direct electrostatic interaction with S4. Instead, their role may be more about, as you say, generating a charge stabilizing hydrophilic environment proximal to the gating charges by attracting water into the VSD structure. Perhaps the way my post is written it emphasizes electrostatics too much and doesn’t emphasize hydrophilicity and water penetration enough. Clearly both are important and related. Additionally, it is very clear from their data the most buried external charge-compensation site (the outer S2 acid) is clearly electrostatic, which completely supports the arguments present in this post.

There are certainly potential caveats to the work of Hoshi and Armstrong and you very nicely point them out. To be devils advocate, the effects they see are from adding 50 μM La3+ – which of course comes along with 150 μM Cl- – to solutions which already contained over 140 mM Na+ and 150 mM Cl-. This is not really comparable to the 10-fold changes in ionic strength done by Islas and Sigworth. Therefore, I would imagine that the ionic strength effects of Debye-screening would be minimal. However, it is certainly possible that strong interactions of La3+ with the phosphate head groups of the lipids could be partially (or wholly) responsible for the changes in gating seen.

In the 2005 Science paper from the von Heijne lab Hessa et al. “conclude that the S4 helix is poised near the threshold of efficient bilayer insertion” and even calculate a positive apparent free energy of translocon-mediated integration. To me this does not indicate that the S4 “readily inserts” or that it can “happily sit in the membrane.” Can it insert, yes, with low efficiency. The fact that, as you say, the membrane likely distorts to accommodate the inserted S4 indicates to me that it is pretty unhappy. Also the fact that Hessa et al. observe that a significant proportion of the S4 helices do not insert into the membrane is also telling. Finally they conclude that, “If one or two of the Arg residues in S4 are partially shielded from direct lipid contact in the intact channel structure, as has been proposed, ΔG_app might be further reduced.” As I demonstrate in this post, the structure shows us how this stabilization is achieved.

You are completely correct in saying that structures do not provide interaction energies and that existing problems with force fields prevent accurate computational deduction of interaction energies. However, the structures do clearly show us which interactions exist and it is now up to us to figure out the experiments that need to be done to determine the specific energies to truly understand their relative importance.

I agree that scientist should avoid becoming attached to their assumptions. There certainly is still a lot of uncertainty and plenty of room for argument ☺

It is well known by anyone who studies these channels that a significant proportion of point mutations results in either no expression or non-functional channels, so clearly a single point mutation can disrupt the entire channel structure. This can also be true for soluble proteins. I am certainly not saying that people shouldn’t do or publish mutagenesis studies, only that we have to be careful when we interpret them. This can be difficult in modern publishing, which encourages and even insists that scientists over sell their findings giving the impression of close-mindedness and being married to assumptions. However, with very few exceptions, most scientists in the field that I have met don’t actually believe their own hype. This problem with scientific publishing is part of the reason I felt the need to start this blog. As per always, the best way to combat close-mindedness in science is to perform more carefully designed well thought out experiments. Anyone who loves science loves being proven wrong. The tricky part is in the proving.