Calculating transition rates and other kinetic quantities from molecular simulations requires knowledge not only of the free energy along the relevant coordinate but also the diffusivity as a function of that coordinate. A variety of methods are currently used to map the free-energy landscape in molecular simulations; however, simultaneous calculation of position-dependent diffusivity is complicated by biasing forces applied with many of these methods. Here, we describe a method to calculate position-dependent diffusivities in simulations including known time-dependent biasing forces, which relies on a previously proposed Bayesian inference scheme. We first apply the method to an explicitly diffusive model, and then to an equilibrium molecular dynamics simulation of liquid water including a position-dependent thermostat, comparing the results to those of an established method. Finally, we test the method on a system of liquid water, where oscillations of the free energy along the coordinate of interest preclude sufficient sampling in an equilibrium simulation. The adaptive biasing force method permits roughly uniform sampling along this coordinate, while the method presented here gives a consistent result for the position-dependent diffusivity, even in a short simulation where the adaptive biasing force is only partially converged.

Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K ؉ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K ؉ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pK a of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pK a, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pK a of charged amino acids within a protein.KCNK channels ͉ molecular simulation ͉ TALK-2 ͉ TASK-2 A ll K ϩ channels contain a highly conserved sequence, the P domain, which forms the selectivity filter and generally six transmembrane ␣-helices. The K ϩ channel pore is formed by four identical subunits, each comprising a P-domain and two of the six transmembrane ␣-helices encircling the ion conduction pathway with a 4-fold symmetry. Structures attached to the pore-forming domains are able to transduce signals, such as changes in transmembrane voltage and intra-or extracellular messages, into gating of the pore (1, 2). Potassium channels of the KCNK superfamily (3, 4) are remarkable in that they possess two P-domains and four ␣-helices in each subunit, form dimers, and are mostly open (''leak channels'') at resting potential. Potassium-selective leaks are fundamental to the function of various cells including nerve, muscle, and epithelia. There are 16 mammalian members to the KCNK family and their gating is variously regulated by free fatty acids, membrane tension, G protein-generated signaling, and extracellular pH. Among KCNK channels gated by extracellular pH, TASK-1 and TASK-3 form a subfamily (TASK) of channels blocked by extracellular protons (5-8). A second subfamily (TALK) of KCNK channels comprises TASK-2, TALK-1, and TALK-2 ¶ , all activated by extracellular alkalinization. TASK-2 participates in ion fluxes necessary for cell volume regulation (10, 11), and its physiological and possible pathological importance has also been highlighted by studies in a TASK-2 knockout mouse (12) that revealed a metabolic acidosis and hypotension caused by renal loss of HCO K ϩ channels of the TASK subfamily are blocked by protons by titration of a histidine (N in TALK channels) residue in the first P domain (6-8), making them responsive to pH in the physiological range. The pH-sensing mechanism ...

Background:The mode of action of PI(4,5)P 2 in TRPV1 is controversial. Results: Positively charged amino acids in the S4-S5 linker and in the TRP box form the PI(4,5)P 2 binding site. Conclusion: PI(4,5)P 2 is a TRPV1 agonist and induces a conformational change of the internal gate. Significance: The molecular nature of the PI(4,5)P 2 binding site in TRPV1 is defined.