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Voltage-gated potassium ion (Kv) channel proteins respond to changes in membrane potential by changing the probability of K + flux through an ion-selective pore. Kv channels from different paralogous and orthologous families have widely varying V 50 values. The voltage-sensing transmembrane helices (S4) of different channels contain four to seven basic residues that are responsible for transducing changes in transmembrane potential into the energy required to shift the equilibrium between the open- and closed-channel conformations. These residues also form electrostatic interaction networks with acidic residues in the S2 and S3 helices that stabilize the open and the closed states to different extents. The length and composition of the extracellular loop connecting the S3 and S4 helices (S3-S4 loop) also shape the voltage response. We describe mutagenesis experiments on the jellyfish (Polyorchis penicillatus) Kv1 family jShak1 channel to evaluate how variants of the S3-S4 loop affect the voltage sensitivity of this channel. In combination with changes in the length and composition of the S3-S4 linker, we mutated a residue on the S2 helix (N227) that in most Kv1 family channels is glutamate (E226 in mouse Kv1.2, E283 in D. melanogaster Shaker). Some individual loop replacement mutants cause major changes in voltage sensitivity, depending on a combination of length and composition. Pairwise combinations of the loop mutations and the S2 mutations interact to yield quantitatively distinct, non-additive changes in voltage sensitivity. We conclude that the S3-S4 loop interacts energetically with the residue at position N227 during the transitions between open and closed states of the channel., (© 2017. Published by The Company of Biologists Ltd.)

Voltage-gated potassium (K(v)) channels work in concert with other ion channels to determine the frequency and duration of action potentials in excitable cells. Little is known about K(v)3 channels from invertebrates, but those that have been characterized generally display slow kinetics. Here, we report the cloning and characterization of jShaw1, the first K(v)3 isolated from a cnidarian, the jellyfish Polyorchis penicillatus, in comparison with mouse K(v)3.1 and K(v)3.2. Using a two-electrode voltage clamp on Xenopus laevis oocytes expressing the channels, we compared steady-state and kinetic properties of macroscopic currents. jShaw1 is fast activating, and opens at potentials approximately 40 mV more hyperpolarized than the mouse K(v)3 channels. There is an inverse relationship between the number of positive charges on the voltage sensor and the half-activation voltage of the channel, contrary to what would be expected with the simplest model of voltage sensitivity. jShaw1 has kinetic characteristics that are substantially different from the mammalian K(v)3 channels, including a much lower sensitivity of early activation rates to incremental voltage changes, and a much faster voltage-dependent transition in the last stages of opening. jShaw1 opening kinetics were affected little by pre-depolarization voltage, in contrast to both mouse channels. Similar to the mouse channels, jShaw1 was half-blocked by 0.7 mmol l(-1) tetraethyl ammonium and 5 mmol l(-1) 4-aminopyridine. Comparison of sequence and functional properties of jShaw1 with the mouse and other reported K(v)3 channels helps to illuminate the general relationship between amino acid sequence and electrophysiological activity in this channel family.

The flow structures produced by the hydromedusae Sarsia tubulosa and Aequorea victoria are examined using direct numerical simulation and Lagrangian coherent structures (LCS). Body motion of each hydromedusa is digitized and input to a CFD program. Sarsia tubulosa uses a jetting type of propulsion, emitting a single, strong, fast-moving vortex ring during each swimming cycle while a secondary vortex of opposite rotation remains trapped within the subumbrellar region. The ejected vortex is highly energetic and moves away from the hydromedusa very rapidly. Conversely, A. victoria, a paddling type hydromedusa, is found to draw fluid from the upper bell surface and eject this fluid in pairs of counter-rotating, slow-moving vortices near the bell margins. Unlike S. tubulosa, both vortices are ejected during the swimming cycle of A. victoria and linger in the tentacle region. In fact, we find that A. victoria and S. tubulosa swim with Strouhal numbers of 1.1 and 0.1, respectively. This means that vortices produced by A. victoria remain in the tentacle region roughly 10 times as long as those produced by S. tubulosa, which presents an excellent feeding opportunity during swimming for A. victoria. Finally, we examine the pressure on the interior bell surface of both hydromedusae and the velocity profile in the wake. We find that S. tubulosa produces very uniform pressure on the interior of the bell as well as a very uniform jet velocity across the velar opening. This type of swimming can be well approximated by a slug model, but A. victoria creates more complicated pressure and velocity profiles. We are also able to estimate the power output of S. tubulosa and find good agreement with other hydromedusan power outputs. All results are based on numerical simulations of the swimming jellyfish.

Voltage sensitivity of voltage-gated potassium channels (VKCs) is a primary factor in shaping action potentials in excitable cells. Variation in the amino acid sequence of the channel proteins is responsible for differences in the voltage range over which the channel opens. Thus, understanding how changes in voltage sensitivity are effected by changes in channel protein sequence illuminates the functional evolution of excitability. The K(V)1-family channel jShak1, from the jellyfish Polyorchis penicillatus, differs from most other K(V)1 channels in ways that are useful for studying the problem of how voltage sensitivity is related to channel sequence. We assessed the contributions of changes in sequence of the S4, voltage sensing, helix and changes in one asparagine residue in the S2 helix, to the relative stability of the open and closed states of the channel. Mutation of the neutral S2 residue (Asn227) to glutamate stabilized the open conformation of the channel. Different modifications of charge and length in S4 favoured either the closed conformation or the open conformation. The interactions between pairs of mutations revealed that some of the S4 mutations alter the conformation of the voltage-sensing domain such that the S4 helix is constrained to be closer to the S2 helix than in the wild-type conformation. These results, taken in conjunction with three-dimensional models of the channel, identify intra-molecular interactions that control the balance between open and closed states. These interactions are likely to be relevant to understanding the functional characteristics of members of this channel family from other organisms.

Cnidocytes were dissociated from the tentacles of the Portuguese Man O'War Physalia physalis using heat treatment, and purified using density centrifugation. Visual observation confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for voltage-gated ion channel subunits using conventional molecular cloning techniques. A variety of channel proteins were identified and full-length sequence obtained for two of them, a Ca(2+) channel beta subunit (PpCa(V)beta) and a Shaker-like K(+) channel (PpK(V)1). The location of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functional properties of these two channel proteins were characterized electrophysiologically using heterologous expression. PpCa(V)beta modulates currents carried by both cnidarian and mammalian alpha(1) subunits although the specifics of the modulation differ. PpK(V)1 produces fast transient outward currents that have properties typical of other Shaker channels. The possible role of these channel proteins in the behavior of cnidocytes is discussed.

Diphyid siphonophores swim using bursts of propulsive jets, which are produced by contractions of a monolayer of subumbrellar myoepithelial fibres lining the nectophore. This swimming behaviour is characterised by successive increases in the force generating the jets during the initial jets of the burst. Action potentials that generate the contractions propagate throughout the myoepithelial layer: both their amplitude and duration successively increase during the first part of the burst. To investigate the ionic mechanism of this action potential augmentation, single myoepithelial cells were enzymatically dissociated and whole-cell voltage clamped. Na+, Ca2+ and K+ currents were recorded under different internal and external salt compositions. The Na+ current was blocked by a relatively high concentration (4 micromol l-1 or higher) of tetrodotoxin (TTX), indicating that the Na+ channel belongs to a group of TTX-resistant Na+ channels. The Ca2+ current was blocked by nifedipine (10 micromol l-1) and Co2+ (5 mmol l-1), indicating that the Ca2+ channel is L-type. The K+ current possessed a unique property of long-lasting inactivation. The K+ current fully inactivated during a depolarisation to +30 mV with a time-constant of approximately 9 ms, and the time constant of recovery from inactivation at -70 mV was 13.2 s. This long-lasting inactivation of the K+ channel was the major factor in the augmentation of both action potentials and contractions of the myoepithelial sheet during the initial part of the burst.

Studies of mitochondrial redox signaling predict that the colonial hydroids Eirene viridula and Podocoryna carnea should respond to manipulations of reactive oxygen species (ROS). Both species encrust surfaces with feeding polyps connected by networks of stolons; P. carnea is more 'sheet-like' with closely spaced polyps and short stolons, while E. viridula is more 'runner-like' with widely spaced polyps and long stolons. Treatment with the chemical antioxidant vitamin C diminishes ROS in mitochondrion-rich epitheliomuscular cells (EMCs) and produces phenotypic effects (sheet-like growth) similar to uncouplers of oxidative phosphorylation. In peripheral stolon tips, treatment with vitamin C triggers a dramatic increase of ROS that is followed by tissue death and stolon regression. The enzymatic anti-oxidant catalase is probably not taken up by the colony but, rather, converts hydrogen peroxide in the medium to water and oxygen. Exogenous catalase does not affect ROS in mitochondrion-rich EMCs, but does increase the amounts of ROS emitted from peripheral stolons, resulting in rapid, runner-like growth. Treatment with exogenous hydrogen peroxide increases ROS levels in stolon tips and results in somewhat faster colony growth. Finally, untreated colonies of E. viridula exhibit higher levels of ROS in stolon tips than untreated colonies of P. carnea. ROS may participate in a number of putative signaling pathways: (1) high levels of ROS may trigger cell and tissue death in peripheral stolon tips; (2) more moderate levels of ROS in stolon tips may trigger outward growth, inhibit branching and, possibly, mediate the redox signaling of mitochondrion-rich EMCs; and (3) ROS may have an extra-colony function, perhaps in suppressing the growth of bacteria.

Redox signaling provides a quick and efficient mechanism for clonal or colonial organisms to adapt their growth and development to aspects of the environment, e.g. the food supply. A 'signature' of mitochondrial redox signaling, particularly as mediated by reactive oxygen species (ROS), can be elucidated by experimental manipulation of the electron transport chain. The major sites of ROS formation are found at NADH dehydrogenase of complex I and at the interface between coenzyme Q and complex III. Inhibitors of complex III should thus upregulate ROS from both sites; inhibitors of complex I should upregulate ROS from the first but not the second site, while uncouplers of oxidative phosphorylation should downregulate ROS from both sites. To investigate the possibility of such redox signaling, perturbations of colony growth and development were carried out using the hydroid Podocoryna carnea. Oxygen uptake of colonies was measured to determine comparable physiological doses of antimycin A(1) (an inhibitor of complex III), rotenone (an inhibitor of complex I) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation). Using these doses, clear effects on colony growth and development were obtained. Treatment with antimycin A(1) results in 'runner-like' colony growth, with widely spaced polyps and stolon branches, while treatment with CCCP results in 'sheet-like' growth, with closely spaced polyps and stolon branches. Parallel results have been obtained previously with azide, an inhibitor of complex IV, and dinitrophenol, another uncoupler of oxidative phosphorylation. Perhaps surprisingly, rotenone produced effects on colony development similar to those of CCCP. Assays of peroxides using 2',7'-dichlorofluorescin diacetate and fluorescent microscopy suggest a moderate difference in ROS formation between the antimycin and rotenone treatments. The second site of ROS formation (the interface between coenzyme Q and complex III) may thus predominate in the signaling that regulates colony development. The fat-rich, brine shrimp diet of these hydroids may be relevant in this context. Acyl CoA dehydrogenase, which catalyzes the first step in the mitochondrial beta-oxidation of fatty acids, carries electrons to coenzyme Q, thus bypassing complex I. These results support a role for redox signaling, mediated by ROS, in colony development. Nevertheless, other redox sensors between complexes I and III may yet be found.