Membrane currents were recorded from isolated fiber cells by using the whole-cell patch clamp technique. A 60-mm tissue culture dish was used as the recording chamber. An Axoclamp 200B patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) or MultiClamp 700A patch clamp amplifier (Molecular Devices) was used to control membrane potential and measure membrane current. The resistance of the patch pipettes was 2.5 to 4 MΩ when filled with standard internal solution. The composition of CsCl internal solutions used in most of the experiments is shown in
Table 1. The final, free calcium concentration of the internal solution was calculated using EQCAL for Windows software (Biosoft, Cambridge, UK). The standard extracellular solution contained (in mM) 140 NaCl, 10 CsCl, 4.7 KCl, 1 MgCl
2, 1 CaCl
2, 5 glucose, and 5 HEPES, with pH adjusted to 7.4 with NaOH. Sodium gluconate external solution was prepared by substituting 140 mM sodium gluconate for equimolar NaCl in the bath solution. The N-methyl-D-glucamine chloride (NMDG-Cl) external solution contained (in mM) 150 NMDG-Cl, 4.7 KCl, 1 MgCl
2, and 1 CaCl
2. The patch pipette was positioned on the cell with a PatchStar micromanipulator (Scientifica, East Sussex, UK). Pulse generation and data acquisition were performed using a personal computer equipped with commercial software (PCLAMP 10; Molecular Devices) and a high-resolution, low-noise digitizer (Digidata 1440A or 1550B; Molecular Devices). The cell was focally perfused with drugs by using a gravity driven, millimanifold applicator (ALA Scientific, Farmingdale, NY, USA) whose tip was brought near and pointed at the cell of study using a manipulator. Solutions flowing over the cell could be changed within a few seconds by using this system. The bath was grounded via a 1-mm-diameter Ag/AgCl wire electrode mounted in a pipette tip filled with 3M KCl agar. Images of fiber cells were acquired via a cooled CCD camera (Coolsnap ES2, Photometrics; Roper Scientific, Tucson, AZ, USA) driven by an imaging program (Nikon Elements AR 4.60; Nikon Instruments, Melville, NY, USA). All the experiments were conducted at room temperature (21–24°C).
For the ion selectivity experiments, the membrane potentials were corrected for liquid-junction potentials after the experiment using the “junction potential calculator” interface (Clampex version 10; Molecular Devices).
Data were analyzed using three different programs (PCLAMP 10 [Molecular Devices], SigmaPlot 11 [Systat Software, Chicago, IL, USA], and Origin 2017 [OriginLab, Northhampton, MA, USA]). Data points were reported as mean ± SEM unless otherwise indicated.