Electrophysiological evidence for light-activated cation transport in calcifying corals

Light has been demonstrated to enhance calcification rates in hermatypic coral species. To date, it remains unresolved whether calcifying epithelia change their ion transport activity during illumination, and whether such a process is mediated by the endosymbiotic algae or can be controlled by the coral host itself. Using a modified Ussing chamber in combination with H + sensitive microelectrode measurements, the present work demonstrates that light triggers the generation of a skeleton positive potential of up to 0.9 mV in the hermatypic coral Stylophora pistillata . This potential is generated by a net flux of cations towards the skeleton and reaches its maximum at blue (450 nm) light. The effects of pharmacological inhibitors targeting photosynthesis 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and anion transport 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) were investigated by pH microelectrode measurements in coral tissues demonstrating a rapid decrease in tissue pH under illumination. However, these inhibitors showed no effect on the electrophysiological light response of the coral host. By contrast, metabolic inhibition by cyanide and deoxyglucose reversibly inhibited the light-induced cation flux towards the skeleton. These results suggest that ion transport across coral epithelia is directly triggered by blue light, independent of photosynthetic activity of algal endosymbionts. Measurements of this very specific and quantifiable physiological response can provide parameters to identify photoreception mechanisms and will help to broaden our understanding of the mechanistic link between light stimulation and epithelial ion transport, potentially relevant for calcification in hermatypic corals.

Stratified Microbial Structure and Activity in Sulfide- and Methane-Producing Anaerobic Sewer Biofilms

ABSTRACTSimultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescencein situhybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera,Desulfobulbus,Desulfomicrobium,Desulfovibrio,Desulfatiferula, andDesulforegula, while about 90% of the MA population belonged to the genusMethanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates.

Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability ( Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I− > NO3− ≅ NO2− > Br− > Cl− > HCO3− > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

Functional effects of 20-HETE on human bronchi: hyperpolarization and relaxation due to BKCa channel activation

Airway smooth muscle (ASM) metabolizes arachidonic acid (AA) through various enzymatic pathways, including cytochrome P-450 (CYP-450) ω-hydroxylase, which leads to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to delineate the mode of action of 20-HETE in human ASM cells. Isometric tension measurements demonstrated that 20-HETE induced a concentration-dependent relaxant effect in ASM on bronchi precontracted with either methacholine or AA. Relaxing effects of 20-HETE on resting tone were prevented by 10 nM iberiotoxin (IbTx), a BKCa channel inhibitor. Microelectrode measurements showed that exogenous additions of 20-HETE (0.1–10 μM) hyperpolarized the membrane potential of human ASM cells. This concentration-dependent electrophysiological effect induced by the eicosanoid was prevented by 10 nM IbTx. Complementary experiments, using the planar lipid bilayer reconstitution technique, demonstrated that 20-HETE activated reconstituted BKCa channels at low free Ca2+ concentrations. Together, these results indicate that 20-HETE-dependent activation of BKCa channels is responsible for the hyperpolarization and controlled relaxation of ASM in human distal bronchi.