TWIK-related Kϩ channel TREK1, a background leak K ϩ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h)TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC 50 value of 180 M, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient ϭ 0.49) and half-of-sites saturation binding stoichiometry (halfreaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and ⌬119 Cterminal deletion mutant (hTREK1 wt -⌬119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000 µmol m s with faster growth and more biomass production when compared to cells at 50 µmol m s light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which is an adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein-pigment complexes which are in agreement with circular dichoism spectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII-LHCII supercomplexes. These results specify that acclimation to high light stress through NPQ mechanism by expression of LHCSR3 and also observed changes in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

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A trace element, iron (Fe) plays a pivotal role in photosynthesis process which in turn mediates the plant growth and productivity. Here, we have focused majorly on the photochemistry of photosystem (PS) II, abundance of proteins, and organization of supercomplexes of thylakoids from Fe-depleted cells in Chlamydomonas reinhardtii. Confocal pictures show that the cell's size has been reduced and formed rosette-shaped palmelloids; however, there is no cell death. Further, the PSII photochemistry was reduced remarkably. Further, the photosynthetic efficiency analyzer data revealed that both donor and acceptor side of PSII were equally damaged. Additionally, the room-temperature emission spectra showed the fluorescence emission maxima increased due to impaired energy transfer from PSII to PSI. Furthermore, the protein data reveal that most of the proteins of reaction center and light-harvesting antenna were reduced in Fe-depleted cells. Additionally, the supercomplexes of PSI and PSII were destabilized from thylakoids under Fe-deficient condition showing that Fe is an important element in photosynthesis mechanism.

Moderately elevated temperatures can induce state transitions in higher plants by phosphorylation of light-harvesting complex II (LHCII). In this study, we exposed unicellular algae Chlamydomonas reinhardtii to moderately elevated temperatures (38 °C) for short period of time in the dark to understand the thylakoid membrane dynamics and state transition mechanism. Here we report that under elevated temperatures (1) LHCII gets phosphorylated similar to higher plants and (2) there is decreased absorption cross section of photosystem II (PSII), whereas (3) there is no change in absorption cross section of photosystem I (PSI) indicating that LHCII trimers are largely disconnected with both photosystems under moderately elevated temperatures and (4) on return to room temperature after elevated temperature treatment there is a formation of state transition complex comprising of PSII-LHCII-PSI. The temperature-induced state transition mechanism also depends on stt7 kinase-like in light-induced state transition. The protein content was stable at the moderately elevated temperature treatment of 40 °C; however, at 45 °C severe downregulation in photosynthetic performance and protein content was observed. In addition to the known changes to photosynthetic apparatus, elevated temperatures can destabilize the PSII-LHCII complex that can result in decreased photosynthetic efficiency in C. reinhardtii. We concluded that the membrane dynamics of light-induced state transitions differs considerably from temperature-induced state transition mechanisms in C. reinhardtii.