Synthesis, Structure and Biological Activity of CIA and CIB, Two α-Conotoxins from the Predation-Evoked Venom of Conus catus

Cone snails produce a fast-acting and often paralyzing venom that is usually injected into their prey or predator through a hypodermic needle-like modified radula tooth. Many diverse compounds are found in their venom including small molecules, peptides and enzymes. However, peptidic toxins called conotoxins (10-40 residues and 2-4 disulfide bonds) largely dominate these cocktails. These disulfide rich toxins are very valuable pharmacological tools for investigating the function of ions channels, G-protein coupled receptors, transporters and enzymes. Here, we report on the synthesis, structure determination and biological activities of two -conotoxins, CIA and CIB, found in the predatory venom of the piscivorous species Conus catus. CIA is a typical 3/5 -conotoxin that blocks the rat muscle type nAChR with an IC50 of 5.7 nM. Interestingly, CIA also inhibits the neuronal rat nAChR subtype 32 with an IC50 of 2.06 M. CIB is a 4/7 -conotoxin that blocks rat neuronal nAChR subtypes, including 32 (IC50 = 128.9 nM) and 7 (IC50 = 1.51 M). High resolution NMR structures revealed typical -conotoxin folds for both peptides. We also investigated the in vivo effects of these toxins on fish, since both peptides were identified in the predatory venom of C. catus. Consistent with their pharmacology, CIA was highly paralytic to zebrafish (ED50 = 110 g/kg), whereas CIB did not affect the mobility of the fish. In conclusion, CIA likely participates in prey capture through muscle paralysis, while the putative ecological role of CIB remains to be elucidated

A Human TREK-1/HEK Cell Line: A Highly Efficient Screening Tool for Drug Development in Neurological Diseases

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model

Mapacalcine protects mouse neurons against hypoxia by blocking cell calcium overload.

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment

Identification and characterization of two zebrafish Twik related potassium channels, Kcnk2a and Kcnk2b

Abstract KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies

Calcium and electrophysiological measures after glutamate stimulation in cortical neurons.

<p>(A) The increase of intracellular calcium was triggered by application of 100 µM of glutamate. The different conditions are summarized under the histogram. <i>Mapa acute</i>, application of 1 µM of mapacalcine immediately before the measure. <i>Mapa pre-incub</i>, application of 1 µM of mapacalcine for 45 min before the measure. <i>Washout</i>, cells were perfused without glutamate. (B–H) Electrophysiological recording of glutamate currents (n = 6 per condition). (B) Typical current trace in control condition and after 100 µM of glutamate application. (C) Typical current trace in control condition and after 100 µM of glutamate application in the presence of 1 µM of mapacalcine. (D) Corresponding histogram of the current density values measured at the glutamate peak. (E) Typical current trace in control condition and after 100 µM of NMDA application. (F) Typical current trace in control condition and after 100 µM of NMDA application in the presence of 1 µM of mapacalcine. (G) Corresponding histogram of the current density values measured at the NMDA peak. (H) Typical current trace in control condition and after 100 µM of glutamate application in the presence of 10 µM of APV and 50 µM of CNQX inhibitors NMDA and AMPA/Kainate receptors, respectively (n = 10). Bars represent the SEM values. ***, p<0.001.</p

Effects of mapacalcine on electrophysiological recordings in cortical neurons in the presence of calcium channel blockers.

<p>(A) Effects of 1 µM of mapacalcine on remaining calcium current after application of 1 µM of ω-conotoxine GVI A, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (B) Effects of 1 µM of mapacalcine on remaining calcium current after application of 250 nM SNX 482, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (C) Effects of 1 µM of mapacalcine on remaining calcium current after application of 75 nM of calcicludine, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (D) % of calcium current inhibition measured at −10 mV by mapacalcine when applied alone (control) or after previous application of the different toxine. Bars represent the SEM values. *, p<0.05, **, p<0.01. n = 10 for each experiment.</p

OGD on cortical neurons: calcium measures.

<p>Typical fluorescence ratio (340nm/380nm) obtained in different conditions. (A) Vehicle without OGD (V), (B) 1 µM mapacalcine without OGD (M), (C) Vehicle immediately after OGD (VaO), (D) Vehicle post-OGD (VpO), (E) Mapacalcine applied for two hours after OGD (MpO), (F) Corresponding histograms. Values are the mean ± SEM (bars). ***, p<0.001.</p

Effects of mapacalcine on electrophysiological recordings in cortical neurons or HEK-293 transfected cells.

<p>(A) Cortical neurons, effects of 1 µM of mapacalcine on remaining calcium current after application of 1 µM of nifedipine, (a) I(pA/pF)  =  f(mV) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (B) Cortical neurons, effects of 1 µM of nifedipine on remaining calcium current after application of 1 µM of mapacalcine, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces recorded at −10 mV, (c) Mean current traces at −10 mV. (C) Comparaison of the % of inhibition of the calcium current measured at −10 mV by mapacalcine when it was applied after or before nifedipine. (D) HEK-293 transfected cells, effects of 1 µM of mapacalcine on Ca_V 1.2 calcium channels, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces. (E) HEK-293 transfected cells, effects of 1 µM of mapacalcine on Ca_V 1.3 calcium channels, (a) I(pA/pF)  =  f(V) curves, (b) Typical current traces. (F) Cortical neurons, effects of a delayed stimulation protocol on mapacalcine calcium channel inhibition. Each circle represent a stimulation cycle consisting in a 0.5 ms pulse from −80 to +10 mV, time between two pulses, 0.5 s. Bars represent the SEM values. *, p<0.05, **, p<0.01. n = 10 for each experiment.</p