The slow rise in fluorescence observed in the wild type was attributed both in part to oxidation of a fraction of PQ pool, in part to redistribution of LHC antenna, increasing the cross section of photosystem II during the transition to state 1. Thus the difference observed in the CSK mutant might be indicative of a perturbation of state 1-state 2 transitions. However, influence of onset and relaxation of the high-energy component of non-photochemical quenching would also contribute to the steady state WZ4002 signal. Yet, we notice that the maximal fluorescence levels determined with a brief saturating pulse immediately before the actinic light 1 is switched off are very similar in the CSK and the wild type. This indicates that, for plants adapted to light 1 conditions, the level of Non Photochemical Quenching and the absorption cross section in the CSK mutant and the wild-type do not differ significantly, as the probability that these two processes compensate exactly for each other is extremely low. However, while the value of the Fm level in the presence of actinic light 2 is slightly lower than Fm1 for the WT these two levels are virtually identical for the CSK mutant. As the changes observed in the WT are consistent with the previous estimate of the mobile LHC during state transitions, this might indicate some impairment of state transition in the CSK deficient mutant. We note that, when light 1 is turned off, the steady state reduction level of PQ pool is again much higher in the CSK mutant compared to the WT. This indicates that the elevated emission under actinic illumination in the CSK mutant, also observed during the first period of light 2 illumination, is not the result of pre-steady state conditions and originates from a more reduced PQ pool in the mutant than in the wild-type. Interestingly, some significant difference between plants grown under standard white light conditions and plants adapted to light 1 are apparent. Excluding the Fv/Fm values, which are essentially the same, it can be seen that: the steady-state levels of fluorescence emission in presence of actinic backgrounds are much more similar in the CSK mutant and WT; the pre-steady state kinetics of the Kautsky transient are faster in CSK mutant than in the WT while the opposite was seen for light 1 adaptation; we observe a significant increase in the level of non-photochemical quenching in CSK mutants with respect to WT, whereas similar levels were observed in light 1 adapted leaves; the Fm1 level is greater than Fm2 in both wildtype and CSK mutants. The increase in nonphotochemical quenching can explain in part the difference in the steady-state level of emission observed in CSK mutant adapted to white light or light 1 conditions, as it will tend to lower the fluorescence emission even in the presence of a more reduced PQ pool, which was more clearly observed in light 1-adapted plants, but it is also apparent when actinic light 1 and light 2 are superimposed, and during the second period of light 2 illumination only. Moreover the apparent rapid kinetic relaxation of the Kautsky transient in the CSK mutant could also be in part due to the onset of NPQ rather than a more rapid attainment of steady-state level.

Each of these channel types plays an essential role in hearing, and all have shown sensitivity to membrane cholesterol content in various cellular models. The large conductance, calcium-activated, ‘BK’-type potassium channels play a variety of roles in the physiological functions of numerous cellular systems. In inner ear hair cells, BK channels are responsible for the temporal precision of sound encoding in mammals. In non-mammals, BK expression and kinetics are responsible for setting the resonant frequency of afferentlyinnervated hair cells. BK channels shape the receptor potential generated by inner hair cells, as evidenced by slowed voltage responses of inner hair cells in mice lacking the pore-forming alpha subunit of BK. The importance of BK channels in audition is underpinned by their expression at the onset of hearing. While cholesterol reportedly modulates BK currents in smooth muscle, glioma, neuronal and endothelial cells, a functional role in auditory hair cells has not been reported previously. In non-mammals, L-type voltage-gated calcium channels serve as the calcium source for BK channels. In all vertebrates, L-type VGCCs also play a key role in neural transmission from the hair cell to the auditory nerve, triggering exocytosis at the basolateral end of the hair cell. Due to high calcium buffering, VGCCs must be in close proximity to BK and to the calcium sensors driving vesicular fusion. Cholesterol inhibits L-type VGCCs in cardiac and coronary myocytes, where cholesterol chelation with MbCD enhances channel activity. However, similarly to the BK channel, the role of cholesterol in Vorinostat modulating VGCCs varies depending upon the particular preparation and cell type studied. Kv and Kir channels counteract depolarization and hyperpolarization of the cell membrane respectively. Kv and Kir channels underlie tuning in the low frequency, apical hair cells of nonmammals. Kv currents play a specialized role in hair cell development, repolarizing the spontaneous action potentials credited with directing the tonotopic organization of the auditory periphery. Cholesterol depletion with MbCD potentiates Kv currents in developing auditory hair cells and abolishes SAPs. Kir currents display sensitivity to cholesterol depletion in a variety of cell types, but the role of cholesterol in modulating Kir in auditory hair cells is unknown. We investigated the effects of the cholesterol depleting drug, MbCD, on the macroscopic currents of the four major ion channel classes responsible for determining the membrane potential of chick auditory hair cells. Depleting cholesterol from the hair cell membrane reduced BK currents while VGCC conductance was increased. There were lesser or absent effects of cholesterol depletion on Kv and Kir currents respectively. Cholesterol staining was most intense at the apical and basolateral ends of the cell and BK channels were identified in cholesterol-enriched microdomains. Our data show that the lipid environment modulates ion channels essential for sound processing and synaptic transmission in the auditory hair cell membrane.