Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery

Modulation of the IKS channel by PIP2 requires two binding sites per monomer

The phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated KV7 channel family, especially the KV7.1 channel. Expressed in the myocardium membrane, KV7.1 forms a complex with KCNE1 auxiliary subunits to generate the IKS current. Here we present molecular models of the transmembrane region of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP2) lipids show that in presence of KCNE1, two PIP2 lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP2 binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP2 in KCNE1 modulation of IKS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels

Studying Kv Channels Function using Computational Methods

International audienceIn recent years, molecular modeling techniques, combined with MD simulations, provided significant insights on voltage-gated (Kv) potassium channels intrinsic properties. Among the success stories are the highlight of molecular level details of the effects of mutations, the unraveling of several metastable intermediate states, and the influence of a particular lipid, PIP2, in the stability and the modulation of Kv channel function. These computational studies offered a detailed view that could not have been reached through experimental studies alone. With the increase of cross disciplinary studies, numerous experiments provided validation of these computational results, which endows an increase in the reliability of molecular modeling for the study of Kv channels. This chapter offers a description of the main techniques used to model Kv channels at the atomistic level

The contribution of lipid peroxidation to membrane permeability in electropermeabilization: A molecular dynamics study

Electroporation or electropermeabilization is a technique that enables transient increase in the cell membrane permeability by exposing cells to pulsed electric field. However, the molecular mechanisms of the long-lived cell membrane permeability, which persists on the minutes time scale after the pulse treatment, remain elusive. Experimental studies have suggested that lipid peroxidation could present a mechanism of this prolonged membrane permeabilization. In this study we make the first important step in quantifying the possible contribution of lipid peroxidation to electropermeabilization. We use free energy calculations to quantify the permeability and conductance of bilayers, containing an increasing percentage of hydroperoxide lipid derivatives, to sodium and chloride ions. We then compare our calculations to experimental measurements on electropermeabilized cells. Our results show that the permeability and conductance increase dramatically by several orders of magnitude in peroxidized bilayers. Yet this increase is not sufficient to reasonably account for the entire range of experimental measurements. Nevertheless, lipid peroxidation might be considered an important mechanism of prolonged membrane permeabilization, if exposure of cells to high voltage electric pulses leads to secondary lipid peroxidation products. Our analysis calls for experimental studies, which will determine the type and amount of lipid peroxidation products in electropermeabilized cell membranes.</p

Voltage-gated ion channel modulation by lipids: Insights from molecular dynamics simulations

AbstractCells commonly use lipids to modulate the function of ion channels. The lipid content influences the amplitude of the ionic current and changes the probability of voltage-gated ion channels being in the active or in the resting states. Experimental findings inferred from a variety of techniques and molecular dynamics studies have revealed a direct interaction between the lipid headgroups and the ion channel residues, suggesting an influence on the ion channel function. On the other hand the alteration of the lipids may in principle modify the overall electrostatic environment of the channel, and hence the transmembrane potential, leading to an indirect modulation, i.e. a global effect. Here we have investigated the structural and dynamical properties of the voltage-gated potassium channel Kv1.2 embedded in bilayers with modified upper or lower leaflet compositions corresponding to realistic biological scenarios: the first relates to the effects of sphingomyelinase, an enzyme that modifies the composition of lipids of the outer membrane leaflets, and the second to the effect of the presence of a small fraction of PIP2, a highly negatively charged lipid known to modulate voltage-gated channel function. Our molecular dynamics simulations do not enable to exclude the global effect mechanism in the former case. For the latter, however, it is shown that local interactions between the ion channel and the lipid headgroups are key-elements of the modulation

Properties of lipid electropores I: Molecular dynamics simulations of stabilized pores by constant charge imbalance

Molecular dynamics (MD) simulations have become a powerful tool to study electroporation (EP) in atomic detail. In the last decade, numerous MD studies have been conducted to model the effect of pulsed electric fields on membranes, providing molecular models of the EP process of lipid bilayers. Here we extend these investigations by modeling for the first time conditions comparable to experiments using long (mu s-ms) low intensity (similar to KV/cm) pulses, by studying the characteristics of pores formed in lipid bilayers maintained at a constant surface tension and subject to constant charge imbalance. This enables the evaluation of structural (size) and electrical (conductance) properties of the pores formed, providing information hardly accessible directly by experiments. Extensive simulations of EP of simple phosphatidylcholine bilayers in 1 M NaCl show that hydrophilic pores with stable radii (1-2.5 nm) form under transmembrane voltages between 420 and 630 mV, allowing for ionic conductance in the range of 6.4-29.5 nS. We discuss in particular these findings and characterize both convergence and size effects in the MD simulations. We further extend these studies in a follow-up paper (Rems et al., Bioelectrochemistry, Submitted), by proposing an improved continuum model of pore conductance consistent with the results from the MD simulations