1. Ionic currents in isolated cultured epithelial cells are studied using whole-cell, tight-seal, patch-clamp recording methods (2).
2. For electrophysiological recording, cells attached to glass cover slips are placed in a shallow chamber and positioned on the stage of a Nikon inverted microscope (Fig. 3).
3. The chamber is superfused with standard external solution from a series of reservoirs and valves designed to provide a regulated gravity-fed flow rate of 1-2 mL/ min. Most drugs and ligands are also applied by bath superfusion from designated reservoirs. Test solutions are applied for a minimum of 5 and usually for 10 complete (1 mL) bath exchanges.
4. For application of test substances by pressure ejection, micropipets (>2 mm in diameter) are positioned 50-100 mm from the cell using a three-dimensional hydraulic manipulator (Narishige Scientific Instrument Lab.) and 2-5 lb/in.2 pressure applied to the back of the micropipet using a Picospritzer II (General Valve Corp.).
5. Patch electrodes are pulled from borosilicate glass with an external diameter of 1.5 mm and an internal diameter of 1.1 mm (#B150-110-10, Sutter Instruments, Novato, CA), using a two stage vertical microelectrode puller (Narishige model PP83). Electrodes have resistances of 3-5 MQ when filled with internal solution and are coated with beeswax to reduce capacitance.
6. The reference electrode is a sealed electrode-salt bridge combination (Dri-ref-2; World Precision Instruments). Offset potentials are nulled using the amplifier circuitry before seals are made on the cells. Liquid junction potentials (LJPs) arising between the bath and the electrode are measured experimentally and defined as the potential of the bath solution with respect to the pipet solution (17). For whole-cell recording, all the data and current-voltage relationships are routinely corrected for LJPs which are calculated as:
where Vm = the membrane potential of the cell and Vp is the pipet potential. To confirm experimentally generated measurements, LJPs are also calculated using a software program (JPCalc, version 2.00; P.H. Barry, Sydney, Australia). For the data shown, LJPs are 9.7 mV for standard low-Cl external Ringers and
K-aspartate pipet solution, and 5.4 mV for low-Cl/low-Ca external and BAPTA intracellular solution.
7. Membrane potential and ionic currents are recorded with an Axopatch 1D amplifier (Axon Instruments) at a temperature of 22-24°C. Currents are filtered with a four-pole low-pass Bessel filter and digitized at a sampling frequency of 5-10 kHz using pCLAMP software, version 6.0 (Axon Instruments). Current and voltage are displayed on a Kikuzui 5040 oscilloscope and on a Gould TA240 chart recorder, and stored on computer disk.
8. Currents are generally elicited in voltage-clamp mode using 500-ms long pulses from a holding potential of -62 mV with steps from -122 to +58 mV. Current amplitude is determined from the steady-state current at the end of the voltage step, and these values are then used to construct current-voltage plots and histograms.
9. In some cases, currents are normalized for cell capacitance and expressed as pA/ pF. The significance of results is determined using Student's i-tests and data are considered significantly different at p < 0.05. Values are expressed as standard error of mean (SEM) where n = number of cells.
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