Whole cell voltage clamp measurements Cells were grown for 1 2 days on 12 mm diameter glass plastic coverslips, which were transferred to an acrylic chamber on the stage of an inverted microscope equipped with Hoffman modulation contrast optics. Cells were superfused at room temperature with a standard external salt solution. Patch pipettes were fabri cated from glass capillaries with a Brown Flaming horizontal micropipette puller. A micromanipulator fixed to the microscope was used to position pipettes. The whole cell patch configurations were obtained by standard patch clamp technique. Voltage clamp currents were mea sured with a patch clamp amplifier with the lowpass, Bessell fil tering set at 5 kHz. Signals from the patch clamp amplifier were fed into a computer via a digital interface and processed by Clampex 8 software.
Ag/AgCl half cells constituted the electrodes, and agar bridges connected the reference electrodes to the bath solution. Series resistances were compensated following whole cell access prior to recordings. Giga ohm seals between pipettes and cell membranes were made with cells perfused with standard external solution. For ICa measurements the cells were perfused with an external solution in which an imper meable cation was substituted for Na , and Ca2 concentra tion was increased. Intracellular Ca2 measurements Cells were loaded with Fura 2/AM by incubating them for 30 min at room temperature with a standard external salt solution con taining 2 uM Fura 2/AM. Cells were then washed with the external salt solution and incubated at 37 C with 5% CO2 for 30 min in the supplemented Claycomb media.
The coverslip was transferred to an acrylic chamber on the microscope stage and washed with the external salt solution AV-951 for 5 minutes before measurements. Temperature was maintained throughout measurements at 37 C by a stage/inline temperature controller Fluorescence was measured with an im aging system consisting of a xenon fiberoptic light source, a filter wheel and a Basler A311F VGA Camera connected to an Olympus IX71inverted fluorescence microscope. The filter wheel and data acquisition were controlled by the InCyte2 soft ware. was determined by interpolation from a standard curve generated by Ca2 calibration buffer kit 2 and Fura 2/K5 salt. After correction for the individual background fluorescence, the ratio of the fluorescence at both excitation wavelengths was monitored simultaneously in 30 40 cells, identified by their fluores cence within a single view field. Images were collected every 3. 3 s. Each slide was perfused with standard exter nal salt solution for 6 min for control measurements, followed by 10 min with the experimental solution.