Journal: Scientific reports

Article Title: Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones.

doi: 10.1038/srep41105

Figure Lengend Snippet: Figure 4. Electrophysiological properties and monovalent cation permeability of CpomOR channels. (a) CpomOrco + OR1 expressing HEK cells respond to the unspecific agonist VUAA3 200 μM generating inward currents in dose dependent manner (a1). Experimental conditions: whole-cell voltage clamp recording; holding potential: −50 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl2, 10 HEPES, pH 7.5. Stimulus intensity was changed by changing stimulus pulse duration. Basal current level was subtracted. VUAA3, 200 μM, applied repeatedly to the extracellular surface of membrane patch in outside-out configuration reversibly increased membrane current noise that can be associated with the activity of ion channels (a2). Experimental conditions: outside-out patch recording; holding potential +50 mV; intracellular solution (mM): KCl 140, EGTA 1, Hepes 10, pH 7.4; extracellular solution (mM): 140 NaCl, 2.0 CaCl2, 10 HEPES, pH 7.5; stimulus: VUAA3, 200 μM. Diagrams in a1 and a2 depict a time course of stimulus presentation. Note: the VUAA3 activated OR channels demonstrate little if any rundown. (b) To estimate the selectivity of the OR channels to monovalent cations we used whole-cell recordings. VUAA3 (200 μM) was added to all extracellular test solutions. Cells were first exposed to NaCl 140 mM solution. Then, the same whole-cell preparation was exposed to a solution in which the Na+ ions were replaced by one of the following cations: Li+, K+, Cs+, Rb+. A series of ramps (50–100, black lines) were used for every solution tested and averaged (color lines). A potential at which current voltage characteristic of VUAA3 activated integral current intercepts current voltage characteristic of basal current was used as a Vr of the OR channel current in a given ion conditions (vertical colour lines). To determine the reversal potential shift (ΔVr), the Vr of the currents obtained in symmetrical Na+ conditions (VrNa+) was subtracted from the Vr obtained, then Na+ was replaced by respective cations (VrX). The ΔVrX means were then used to estimate the permeability ratios (PX+/PNa+). The permeability ratio sequence for some inorganic monovalent cations (PX+/PNa+) was: Rb+ (2 ± 0.12) > K+ (1.37 ± 0.03) ≥ Cs+ (1.36 ± 0.03) ~ Na+ > Li+ (0.93 ± 0.06). Experimental conditions: whole-cell voltage clamp recording; holding potential: −60 mV; intracellular solution (mM): NaCl 140, EGTA 0.5, Hepes 10, pH 7.4; extracellular solution: varied in accordance with the above description.

Article Snippet: HEK293T cells were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL Penicillin/Streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Permeability, Expressing, Membrane, Activity Assay, Sequencing