PMC

Activation of ArchT in transfected cells acidifies the external pH. A, calibration of an external pH-selective microelectrode filled with an H⁺-ionophore 1 mixture. A reference electrode was placed close to the pH microelectrode. Voltages were acquired with a high impedance dual channel differential electrometer. The slope of the curve corresponding to the Nernst potential for H⁺ is shown: −58 mV/pH unit. B, examples of cell surface pH recorded from CHO cells transfected with pcDNA3.1-ArchT-GFP. Traces are voltage changes recorded with the external pH microelectrode positioned close to the cell surface of a cell illuminated with light 580 ± 30 nm, power 3 mW/mm². Yellow shade represents the illumination time. Left of the traces is a pH scale constructed from the calibration shown in A. Illumination induces an external pH change of ∼0.2 pH unit. Data were acquired via a digitizer (iWorx/214) connected to a PC computer, visualized, and recorded using LabScribe software.

Activation of ASIC1 by illumination of ArchT in cultured cells. A, fluorescent image of cells transfected with ArchT-GFP. B, whole cell currents of a representative ArchT-expressing cell evoked by illumination with 580 ± 30 nm light (3 mW/mm²) for 0.4-s pulses (orange bars). The magnitude of the outward currents decreased as the pH of the bath solution was decreased from 7.4 to 6.5, 4.0, and 2.5. Membrane voltage was held at −40 mV. C, illumination of a cell co-expressing ArchT and human ASIC1a evoking a biphasic current. Immediately after photoactivation of the outward current there is a transient component of inward current that decays in subsequent light pulses (second and third traces shown in gray). D, protocol identical to that shown in B but with 100 μm amiloride in the bath eliminating the inward component of the currents. E, after washout of amiloride, rapid application of three consecutive pH 6.5 stimuli evoking only inward currents with properties distinctive of hASIC1a (first trace in red with second and third shown in gray).

Photoactivation of endogenous ASIC1a in brain slices. A, representative responses of pyramidal neurons from mouse visual cortex show expression of ArchT in astrocytes. Optical stimulation of astrocytes with amber light supplied by a 590-nm LED (orange trace below voltage recordings) depolarized the membrane potential of neighboring neurons detected by an intracellular electrode. Increasing the light stimulus, i.e. lengthening the pulses from 12 to 50 ms, evoked larger depolarizations and triggered action potentials. The response was completely blocked in the presence of the ASIC1a-specific inhibitor PcTx1. B, a similar experiment conducted in slices previously injected with adenovirus expressing only GFP did not respond to illumination. C, current traces of pyramidal cells from the same brain slice but obtained in the voltage clamp configuration were evoked by rapid changes in the external pH solution from 7.3 to pH 6.9 or pH 5.0. PcTx1 in the bath prevented proton-induced currents. Time between stimuli was 60 s. D, depolarization and train of action potentials are attenuated when the stimulus is applied at short intervals. Full recovery was observed at intervals ≥50 s.

Expression of ArchT-GFP in mouse astrocytes. A, diagram of the expression cassette. The compact glial fibrillary acidic protein promoter GfaABC1D (mGFAP) and the minimal core promoter derived from the human cytomegalovirus (mCMV), both shown in red, are in opposite orientations and are followed by the coding regions of the two proteins to be expressed: the enhancer chimeric protein GAL4BD-NFκBp65 that binds to five repeats of the GAL4 binding sequence (5×GAL4 BS) and ArchT-EGFP. WPRE, woodchuck hepatitis post-transcriptional regulatory element for enhancement of gene expression. LITR and RITR, left and right inverted terminal repeats, respectively. Shown below are the same adenoviral construct but expressing only GFP. B, primary mouse astrocytes grown in culture transfected with adenovirus expressing ArchT-GFP driven by GFAP promoter. Endogenous GFAP was stained with a monoclonal antibody conjugated with Alexa Fluor 594. C, confocal images of a 20-μm-thick brain slice of brain cortex fixed with 4% paraformaldehyde prepared 2 weeks after stereotaxic injection of ArchT-GFP adenoviral vector. Slices were stained with monoclonal anti-GFAP conjugated with Alexa Fluor 594. Images were obtained in an inverted Zeiss Confocal Microscope (LSM 780). The three channels (DAPI, GFP, and Alexa Fluor 594) were scanned separately and merged using Zen software.