Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SKF 96356, and thapsigargin but not by preinjection of heparin, 8-NH2- cyclic ADP-ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin-A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current that requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC.

NAADP activates a Ca2+ current that is dependent on F-actin cytoskeleton

Future brain-machine interfaces, prosthetics, and intelligent soft robotics will require integrating artificial neuromorphic devices with biological systems. Due to their poor biocompatibility, circuit complexity, low energy efficiency, and operating principles fundamentally different from the ion signal modulation of biology, traditional Silicon-based neuromorphic implementations have limited bio-integration potential. Here, we report the first organic electrochemical neurons (OECNs) with ion-modulated spiking, based on all-printed complementary organic electrochemical transistors. We demonstrate facile bio-integration of OECNs with Venus Flytrap (Dionaea muscipula) to induce lobe closure upon input stimuli. The OECNs can also be integrated with all-printed organic electrochemical synapses (OECSs), exhibiting short-term plasticity with paired-pulse facilitation and long-term plasticity with retention >1000 s, facilitating Hebbian learning. These soft and flexible OECNs operate below 0.6 V and respond to multiple stimuli, defining a new vista for localized artificial neuronal systems possible to integrate with bio-signaling systems of plants, invertebrates, and vertebrates. 

/pdf/organic-electrochemical-neurons-and-synapses-with-ion-2d8q8ac4.pdf

Organic electrochemical neurons and synapses with ion mediated spiking

NAADP triggers the fertilization potential in starfish oocytes

During skeletal muscle excitation-contraction (EC) coupling, membrane depolarizations activate the sarcolemmal voltage-gated L-type Ca(2+) channel (Ca(V)1.1). Ca(V)1.1 in turn triggers opening of the sarcoplasmic Ca(2+) release channel (RyR1) via interchannel protein-protein interaction to release Ca(2+) for myofibril contraction. Simultaneously to this EC coupling process, a small and slowly activating Ca(2+) inward current through Ca(V)1.1 is found in mammalian skeletal myotubes. The role of this Ca(2+) influx, which is not immediately required for EC coupling, is still enigmatic. Interestingly, whole-cell patch clamp experiments on freshly dissociated skeletal muscle myotubes from zebrafish larvae revealed the lack of such Ca(2+) currents. We identified two distinct isoforms of the pore-forming Ca(V)1.1alpha(1S) subunit in zebrafish that are differentially expressed in superficial slow and deep fast musculature. Both do not conduct Ca(2+) but merely act as voltage sensors to trigger opening of two likewise tissue-specific isoforms of RyR1. We further show that non-Ca(2+) conductivity of both Ca(V)1.1alpha(1S) isoforms is a common trait of all higher teleosts. This non-Ca(2+) conductivity of Ca(V)1.1 positions teleosts at the most-derived position of an evolutionary trajectory. Though EC coupling in early chordate muscles is activated by the influx of extracellular Ca(2+), it evolved toward Ca(V)1.1-RyR1 protein-protein interaction with a relatively small and slow influx of external Ca(2+) in tetrapods. Finally, the Ca(V)1.1 Ca(2+) influx was completely eliminated in higher teleost fishes.

https://www.pnas.org/content/pnas/107/12/5658.full.pdf

Non–Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling

The functional separation between skeletal and cardiac muscles, which occurs at the threshold between vertebrates and invertebrates, involves the evolution of separate contractile and control proteins for the two types of striated muscles, as well as separate mechanisms of contractile activation. The functional link between electrical excitation of the surface membrane and activation of the contractile material (known as excitation-contraction [e-c] coupling) requires the interaction between a voltage sensor in the surface membrane, the dihydropyridine receptor (DHPR), and a calcium release channel in the sarcoplasmic reticulum, the ryanodine receptor (RyR). Skeletal and cardiac muscles have different isoforms of the two proteins and present two structurally and functionally distinct modes of interaction. We use structural clues to trace the evolution of the dichotomy from a single, generic type of e-c coupling to a diversified system involving a novel mechanism for skeletal muscle activation. Our results show that a significant structural transition marks the protochordate to the Craniate evolutionary step, with the appearance of skeletal muscle-specific RyR and DHPR isoforms.

/pdf/evolution-of-skeletal-type-e-c-coupling-a-novel-means-of-5grg1fxiai.pdf

Evolution of skeletal type e–c coupling: a novel means of controlling calcium delivery

The leaves of the carnivorous plant Venus flytrap, Dionaea muscipula (Dionaea) close rapidly to capture insect prey. The closure response usually requires two successive mechanical stimuli to sensory hairs on the leaf blade within approximately 30 s (refs. 1–4). An unknown biological system in Dionaea is thought to memorize the first stimulus and transduce the signal from the sensory hair to the leaf blade2. Here, we link signal memory to calcium dynamics using transgenic Dionaea expressing a Ca2+ sensor. Stimulation of a sensory hair caused an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) starting in the sensory hair and spreading to the leaf blade. A second stimulus increased [Ca2+]cyt to an even higher level, meeting a threshold that is correlated to the leaf blade closure. Because [Ca2+]cyt gradually decreased after the first stimulus, the [Ca2+]cyt increase induced by the second stimulus was insufficient to meet the putative threshold for movement after about 30 s. The Ca2+ wave triggered by mechanical stimulation moved an order of magnitude faster than that induced by wounding in petioles of Arabidopsis thaliana5 and Dionaea. The capacity for rapid movement has evolved repeatedly in flowering plants. This study opens a path to investigate the role of Ca2+ in plant movement mechanisms and their evolution. A transgenic Venus flytrap expressing a fluorescent calcium sensor allows real-time live quantification of calcium waves triggered by sensory hair movement. The study suggests that calcium levels represent the molecular basis for the memory effect that requires two stimulations within 30 s.

Calcium dynamics during trap closure visualized in transgenic Venus flytrap.

Hagfishes are regarded as the most primitive living craniates. Excitation-contraction (E-C) coupling mechanisms were studied in skeletal and caudal heart muscle fibres of the hagfish Eptatretus burgeri. In white (fast) skeletal muscle fibres from the musculus tubulatus, force generation in response to electrical stimulation was maintained in nominally Ca(2+) free artificial seawater (ASW) (0Ca(2+)-ASW) containing 10 mmol l(-1) Co(2+) (a blocker of Ca(2+) currents). Similarly, in red (slow) fibres from parietal muscle bathed in 0Ca(2+)-ASW containing 10 mmol l(-1) Co(2+), force generation occurred in association with K(+) depolarisation when the external K(+) concentration was increased to 100 mmol l(-1). Therefore, external Ca(2+) is not required for muscle contraction. Hence, both white and red fibres possess the function of depolarisation-induced Ca(2+)-release from intracellular Ca(2+) stores. This function is the same as in the skeletal muscle of all other vertebrates. In caudal heart muscle fibres, twitches in response to electrical stimuli were maintained in 0Ca(2+)-ASW containing 30 mmol l(-1) Co(2+). In fibres loaded with fluo-3 bathed in 0Ca(2+)-ASW containing 30 mmol l(-1) Co(2+), an increase in the intracellular free Ca(2+) level associated with K(+) depolarisation was observed after the external K(+) concentration was increased to 100 mmol l(-1). Thus E-C coupling in the caudal heart muscle is also of the vertebrate skeletal muscle type.

Excitation-contraction coupling in skeletal and caudal heart muscle of the hagfish Eptatretus burgeri Girard.

Ionic currents from Schwann cells isolated enzymatically from the giant axons of the squids Loligo forbesi, Loligo vulgaris and Loligo bleekeri were compared with those obtained in situ. Macroscopic and single channel ionic currents were recorded using whole-cell voltage and patch clamp. In the whole-cell configuration, depolarisation from negative holding potentials evoked two voltage-dependent currents, an inward current and a delayed outward current. The outward current resembled an outwardly rectifying K+ current and was activated at −40 mV after a latent period of 5-20 ms following a step depolarisation. The current was reduced by externally applied nifedipine, Co2+ or quinine, was not blocked by addition of apamin or charibdotoxin and was insensitive to externally applied l-glutamate or acetylcholine. The voltage-gated inward current was activated at −40 mV and was identified as an L-type calcium current sensitive to externally applied nifedipine. Schwann cells were impaled in situ in split-open axons and voltage clamped using discontinuous single electrode voltage clamp. Voltage dependent outward currents were recorded that were kinetically identical to those seen in isolated cells and that had similar current-voltage relations. Single channel currents were recorded from excised inside-out patches. A single channel type was observed with a reversal potential close to the equilibrium potential for K+ (EK) and was therefore identified as a K+ channel. The channel conductance was 43.6 pS when both internal and external solutions contained 150 mm K+. Activity was weakly dependent on membrane voltage but sensitive to the internal Ca2+ concentration. Activity was insensitive to externally or internally applied l-glutamate or acetylcholine. The results suggest that calcium channels and calcium-activated K+ channels play an important role in the generation of the squid Schwann cell membrane potential, which may be controlled by the resting intracellular Ca2+ level.

Ionic currents in isolated and in situ squid Schwann cells

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 + , Ca 2 + and K + currents were recorded under different internal and external salt compositions. The Na + current was blocked by a relatively high concentration (4 μmol l - 1 or higher) of tetrodotoxin (TTX), indicating that the Na + channel belongs to a group of TTX-resistant Na + channels. The Ca 2 + current was blocked by nifedipine (10 μmol l - 1 ) and Co 2 + (5 mmol l - 1 ), indicating that the Ca 2 + 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 ∼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.

/pdf/long-lasting-potassium-channel-inactivation-in-myoepithelial-rh5hf8dis6.pdf

Izuo Tsutsui

Papers

Calcium dynamics during trap closure visualized in transgenic Venus flytrap.

Excitation-contraction coupling in skeletal and caudal heart muscle of the hagfish Eptatretus burgeri Girard.

Ionic currents in isolated and in situ squid Schwann cells

Long-lasting potassium channel inactivation in myoepithelial fibres is related to characteristics of swimming in diphyid siphonophores