Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control.

In the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp.

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

Orthosteric muscarinic receptor activation by the insect repellent IR3535 opens new prospects in insecticide-based vector control.

The insect repellent IR3535 is one of the important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika. Using a multidisciplinary approach, we propose the development of an innovative insecticide-based vector control strategy using an unexplored property of IR3535. We have demonstrated that in insect neurosecretory cells, very low concentration of IR3535 induces intracellular calcium rise through cellular mechanisms involving orthosteric/allosteric sites of the M1-muscarinic receptor subtype, G protein ßγ subunits, background potassium channel inhibition generating depolarization, which induces voltage-gated calcium channel activation. The resulting internal calcium concentration elevation increases nicotinic receptor sensitivity to the neonicotinoid insecticide thiacloprid. The synergistic interaction between IR3535 and thiacloprid contributes to significantly increase the efficacy of the treatment while reducing concentrations. In this context, IR3535, used as a synergistic agent, seems to promise a new approach in the optimization of the integrated vector management for vector control.

Synergistic agent and intracellular calcium, a successful partnership in the optimization of insecticide efficacy.

Integrated Pest Management and Integrated Vector Management worldwide are developed in agriculture and public health to counteract and limit the exponential increasing development of insect resistance to insecticides. However, facing the predominance of some resistant populations, new strategies are urgently needed to target resistant insects. An innovative approach lies in the optimization of commonly used insecticides when combined with chemical or biological synergistic agents. By an increase of intracellular calcium concentration followed by activation of calcium-dependant signalling pathways, the synergistic agents are able to indirectly increase target sites sensitivity to insecticide by inducing conformational change. The synergistic agents are of great interest in optimizing the efficacy of insecticides and in overcoming resistance mechanisms.

Modulation of Low-Voltage-Activated Inward Current Permeable to Sodium and Calcium by DARPP-32 Drives Spontaneous Firing of Insect Octopaminergic Neurosecretory Cells.

Identification of the different intracellular pathways that control phosphorylation/dephosphorylation process of ionic channels represents an exciting alternative approach for studying the ionic mechanisms underlying neuronal pacemaker activity. In the central nervous system of the cockroach Periplaneta americana, octopaminergic neurons, called dorsal unpaired median (DUM; DUM neurons), generate spontaneous repetitive action potentials. Short-term cultured adult DUM neurons isolated from the terminal abdominal ganglion (TAG) of the nerve cord were used to study the regulation of a tetrodotoxin-sensitive low-voltage-activated (LVA) channel permeable to sodium and calcium (Na/Ca), under whole cell voltage- and current-clamp conditions. A bell-shaped curve illustrating the regulation of the amplitude of the maintained current vs. [ATP]i was observed. This suggested the existence of phosphorylation mechanisms. The protein kinase A (PKA) inhibitor, H89 and elevating [cyclic adenosine 3', 5' monophosphate, cAMP]i, increased and decreased the current amplitude, respectively. This indicated a regulation of the current via a cAMP/PKA cascade. Furthermore, intracellular application of PP2B inhibitors, cyclosporine A, FK506 and PP1/2A inhibitor, okadaic acid decreased the current amplitude. From these results and because octopamine (OA) regulates DUM neuron electrical activity via an elevation of [cAMP]i, we wanted to know if, like in vertebrate dopaminergic neurons, OA receptor (OAR) stimulation could indirectly affect the current via PKA-mediated phosphorylation of Dopamine- and cAMP-regulated Phosphoprotein-32 (DARPP-32) known to inhibit PP1/2A. Experiments were performed using intracellular application of phospho-DARPP-32 and non-phospho-DARPP-32. Phospho-DARPP-32 strongly reduced the current amplitude whereas non-phospho-DARPP-32 did not affect the current. All together, these results confirm that DARPP-32-mediated inhibition of PP1/2A regulates the maintained sodium/calcium current, which contributes to the development of the pre-depolarizing phase of the DUM neuron pacemaker activity.

Dysfunction of the Voltage-Gated K+ Channel ß2 Subunit in a Familial Case of Brugada Syndrome.

BACKGROUND: The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN5A, the molecular mechanisms underlying this condition are still largely unknown. METHODS AND RESULTS: We combined whole-exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB2, which encodes the voltage-gated K(+) channel ß2-subunit (Kvß2-R12Q). Kvß2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K(+) channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB2 variants by using cellular electrophysiology and biochemistry. Patch-clamp experiments performed in COS-7 cells expressing both Kv4.3 and Kvß2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvß2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvß2-R12Q were significantly increased in comparison with wild-type Kvß2. CONCLUSIONS: Altogether, our results indicate that Kvß2 dysfunction can contribute to the Brugada electrocardiographic pattern.

Improvements in purification of silver nanowires by decantation and fabrication of flexible transparent electrodes. Application to capacitive touch sensors.

Transparent flexible electrodes made of metallic nanowires, and in particular silver nanowires (AgNWs), appear as an extremely promising alternative to transparent conductive oxides for future optoelectronic devices. Though significant progresses have been made the last few years, there is still some room for improvement regarding the synthesis of high quality silver nanowire solutions and fabrication process of high performance electrodes. We show that the commonly used purification process can be greatly simplified through decantation. Using this process it is possible to fabricate flexible electrodes by spray coating with sheet resistance lower than 25 Ω sq⁻¹ at 90% transparency in the visible spectrum. These electrodes were used to fabricate an operative transparent flexible touch screen. To our knowledge this is the first reported AgNW based touch sensor relying on capacitive technology.