The purified mechanosensitive channel TREK-1 is directly sensitive to membrane tension.

Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K(+) channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.

Is the redox state of the ci heme of the cytochrome b6f complex dependent on the occupation and structure of the Qi site and vice versa?

Oxidoreductases of the cytochrome bc(1)/b(6)f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b(6)f has revealed the presence in the Q(i) site of an atypical c-type heme, heme c(i). Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the c(i) heme and its interaction with the Q(i) site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe(40) from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the c(i) heme midpoint potential (from +100 mV to -200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b(6)f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis.

b6f-Associated chlorophyll: structural and dynamic contribution to the different cytochrome functions.

Cytochromes bc1/b6f complexes catalyze electron transfer from lipid- to water-soluble carriers in both the respiratory and photosynthetic processes. They contain several common redox cofactors, while a chlorophyll a molecule, the function of which is still enigmatic, is only present in b b6f-type complexes. In this work, we describe a mutagenesis approach aimed at characterizing the role of this pigment. Mutants of the binding pocket were constructed to obtain cytochrome (cyt) b6f f complexes altered in chlorophyll position and/or stability. On the basis of a combined biochemical and functional analysis, we conclude that the chlorophyll plays a major structural role in the complex. Moreover, the chlorophyll and its binding pocket may also be implicated in the regulation of photosynthetic state transitions, a function that is specific to cyt b6f complexes.

Why is it so difficult to construct Q(i) site mutants in Chlamydomonas reinhardtii?

The cytochrome b(6)f complex catalyses electron transfer from plastoquinol to a hydrosoluble acceptor (plastocyanin), while building up an electrochemical proton gradient. Oxidation and reduction of plastoquinol occur respectively at the Q(o) site (exposed on the luminal side of the thylakoid membrane) and at the Q(i) site (facing the stroma). The discovery of an additional c'-type heme in the Q(i) site has cast a new light on the difficulties previously encountered to obtain mutants at this site. In this work, we critically examine our unsuccessful attempts to obtain Q(i) site mutants based on sequence and structure homology between cytochrome b(6)f and bc(1) complexes.

DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2.

The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Competing ligands such as mannan and an antibody directed against the carbohydrate recognition domains (CRD) abrogated binding. While no E2 interaction with distant monomeric CRDs on biosensor chips could be detected, binding is observed if CRDs are closely seeded (Kd = 48 nm) and if the CRD is part of the oligomeric-soluble extracellular domain of DC-SIGN (Kd = 30 nm). The highest affinity is seen for plasma membrane-expressed DC-SIGN and L-SIGN (Kd = 3 and 6 nm, respectively). These results indicate that several high-mannose N-glycans in a structurally defined cluster on E2 bind to several subunits of the oligomeric lectin CRD. High affinity interaction of viral glycoproteins with oligomeric lectins might represent a strategy by which HCV targets to and concentrates in the liver and infects dendritic cells.