Ca²+ measurements.

NHC cells were plated onto glass coverslips 24 hours prior to imaging. Cells were then loaded with 6 µM Fluo-4/AM (Invitrogen, Eugene, OR) for 30 minutes at 37° C. Once transferred to a custom-built perfusion chamber on the stage of a Zeiss LSM 710 confocal microscope (Carl Zeiss, Inc, Thornwood, NY), cells were perfused with HEPES buffer (16) while stimulated with 1 µM adenosine triphosphate (ATP). Ca²+ signaling was monitored in these cells by exciting at 488 nm while collecting emitted light above 505 nm. Normalized amplitudes of the ATP-induced Ca²⁺ signals were extracted with Image J software and plotted as previously described (17). For flash photolysis of caged InsP3, cells were transfected with a rat mCherry-ITPR3 plasmid (a gift from David Yule, University of Rochester Medical Center) and 48 hours later incubated with 1 μM caged InsP3 (Bio-Techne, Minneapolis, MN) and 6 µM Fluo-4/AM for 30 minutes at 37° C. Cells were then imaged on a Bruker Opterra II Swept field confocal microscope (Middleton, WI) using 488 nm excitation and collection above 505 nm. Uncaging was achieved by a brief pulse of 405 nm laser line on individual cells (36 µs/pixel). Changes in fluorescence were normalized by baseline levels and used to calculated the rise time of Ca²⁺ signals, which is the time required for the signal to increase from 20% to 80% of the maximum response.