Effect of FMRFamide on voltage-dependent currents in identified centrifugal neurons of the optic lobe of the cuttlefish, Sepia officinalis

Whole-cell patch-clamp recordings from identified centrifugal neurons of the optic lobe in a slice preparation allowed the characterization of five voltage-dependent currents; two outward and three inward currents. The outward currents were; the 4-aminopyridine-sensitive transient potassium or A-current (IA), the TEA-sensitive sustained current or delayed rectifier (IK). The inward currents were; the tetrodotoxin sensitive transient current or sodium current (INa). The second is the cobalt- and cadmium-sensitive sustained current which is enhanced by barium and blocked by the dihydropyridine antagonist, nifedipine suggesting that it could be the L-type calcium current (ICaL). Finally, another transient inward current, also carried by calcium, but unlike the L-type, this current is activated at more negative potentials and resembles the low-voltage-activated or T-type calcium current (ICaT) of other preparations. Application of the neuropeptide FMRFamide caused a significant attenuation to the peak amplitude of both sodium and sustained calcium currents without any apparent effect on the transient calcium current. Furthermore, FMRFamide also caused a reduction of both outward currents in these centrifugal neurons. The fact that FMRFamide reduced the magnitude of four of five characterized currents could suggest that this neuropeptide may act as a strong inhibitory agent on these neurons

Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH

Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH

The requirement for external carbonic anhydrase in diatoms is influenced by the supply and demand for dissolved inorganic carbon

Photosynthesis by marine diatoms contributes significantly to the global carbon cycle. Due to the low concentration of CO2 in seawater, many diatoms use extracellular carbonic anhydrase (eCA) to enhance the supply of CO2 to the cell surface. While much research has investigated how the requirement for eCA is influenced by changes in CO2 availability, little is known about how eCA contributes to CO2 supply following changes in the demand for carbon. We therefore examined how changes in photosynthetic rate influence the requirement for eCA in three centric diatoms. Modeling of cell surface carbonate chemistry indicated that diffusive CO2 supply to the cell surface was greatly reduced in large diatoms at higher photosynthetic rates. Laboratory experiments demonstrated a trend of an increasing requirement for eCA with increasing photosynthetic rate that was most pronounced in the larger species, supporting the findings of the cellular modeling. Microelectrode measurements of cell surface pH and O2 demonstrated that individual cells exhibited an increased contribution of eCA to photosynthesis at higher irradiances. Our data demonstrate that changes in carbon demand strongly influence the requirement for eCA in diatoms. Cell size and photosynthetic rate will therefore be key determinants of the mode of dissolved inorganic carbon uptake

A Novel Single-Domain Na+-Selective Voltage-Gated Channel in Photosynthetic Eukaryotes

The evolution of Na+-selective four-domain voltage-gated channels (4D-Navs) in animals allowed rapid Na+-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signaling. While bacteria encode single-domain Na+-selective voltage-gated channels (BacNav), they typically exhibit much slower kinetics than 4D-Navs, and are not thought to have crossed the prokaryote–eukaryote boundary. As such, the capacity for rapid Na+-selective signaling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus flytrap (Dionaea muscipula) where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na+-selective, single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores, Emiliania huxleyi and Scyphosphaera apsteinii. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and isoform-specific sensitivity to the highly selective 4D-Nav blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na+-based signaling in eukaryotes is not restricted to animals or to the presence of 4D-Navs. The EukCatB channels therefore represent an independent evolution of fast Na+-based electrical signaling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae

Alternative Mechanisms for Fast Na+/Ca2+ Signaling in Eukaryotes via a Novel Class of Single-Domain Voltage-Gated Channels

This is the final version. Available from Elsevier via the DOI in this record.Rapid Na+/Ca2+-based action potentials govern essential cellular functions in eukaryotes, from the motile responses of unicellular protists, such as Paramecium [1, 2], to complex animal neuromuscular activity [3]. A key innovation underpinning this fundamental signaling process has been the evolution of four-domain voltage-gated Na+/Ca2+ channels (4D-Cavs/Navs). These channels are widely distributed across eukaryote diversity [4], albeit several eukaryotes, including land plants and fungi, have lost voltage-sensitive 4D-Cav/Navs [5, 6, 7]. Because these lineages appear to lack rapid Na+/Ca2+-based action potentials, 4D-Cav/Navs are generally considered necessary for fast Na+/Ca2+-based signaling [7]. However, the cellular mechanisms underpinning the membrane physiology of many eukaryotes remain unexamined. Eukaryotic phytoplankton critically influence our climate as major primary producers. Several taxa, including the globally abundant diatoms, exhibit membrane excitability [8, 9, 10]. We previously demonstrated that certain diatom genomes encode 4D-Cav/Navs [4] but also proteins of unknown function, resembling prokaryote single-domain, voltage-gated Na+ channels (BacNavs) [4]. Here, we show that single-domain channels are actually broadly distributed across major eukaryote phytoplankton lineages and represent three novel classes of single-domain channels, which we refer collectively to as EukCats. Functional characterization of diatom EukCatAs indicates that they are voltage-gated Na+- and Ca2+-permeable channels, with rapid kinetics resembling metazoan 4D-Cavs/Navs. In Phaeodactylum tricornutum, which lacks 4D-Cav/Navs, EukCatAs underpin voltage-activated Ca2+ signaling important for membrane excitability, and mutants exhibit impaired motility. EukCatAs therefore provide alternative mechanisms for rapid Na+/Ca2+ signaling in eukaryotes and may functionally replace 4D-Cavs/Navs in pennate diatoms. Marine phytoplankton thus possess unique signaling mechanisms that may be key to environmental sensing in the oceans.European Research CouncilNS

A Voltage-Gated H+ Channel Underlying pH Homeostasis in Calcifying Coccolithophores

Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths) in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO3− as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan Hv1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the Hv1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate use of H+ derived from calcification for intracellular CO2 generation. Furthermore, the presence of Hv1 class ion channels in a wide range of extant eukaryote groups indicates they evolved in an early common ancestor

Functional Changes in the Snail Statocyst System Elicited by Microgravity

BACKGROUND: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. METHODOLOGY/PRINCIPAL FINDINGS: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. CONCLUSIONS/SIGNIFICANCE: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space