Endogenous cytosolic Ca2+ buffering is necessary for TRPM4 activity in cerebral artery smooth muscle cells

The melastatin transient receptor potential (TRP) channel, TRPM4, is a critical regulator of smooth muscle membrane potential and arterial tone. Activation of the channel is Ca2+-dependent, but prolonged exposures to high global Ca2+ causes rapid inactivation under conventional whole-cell patch clamp conditions. Using amphotericin B perforated whole cell patch clamp electrophysiology, which minimally disrupts cytosolic Ca2+ dynamics, we recently showed that Ca2+ released from 1,2,5-triphosphate receptors (IP3R) on the sarcoplasmic reticulum (SR) activates TRPM4 channels, producing sustained transient inward cation currents (TICCs). Thus, Ca2+-dependent inactivation of TRPM4 may not be inherent to the channel itself but rather is a result of the recording conditions. We hypothesized that under conventional whole-cell configurations, loss of intrinsic cytosolic Ca2+ buffering following cell dialysis contributes to inactivation of TRPM4 channels. With the inclusion of the Ca2+ buffers ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 10 mM) or bis-ethane-N,N,N′,N′-tetraacetic acid (BAPTA, 0.1 mM) in the pipette solution, we mimic endogenous Ca2+ buffering and record novel, sustained whole-cell TICC activity from freshly-isolated cerebral artery myocytes. Biophysical properties of TICCs recorded under perforated and whole-cell patch clamp were nearly identical. Furthermore, whole-cell TICC activity was reduced by the selective TRPM4 inhibitor, 9-phenanthrol, and by siRNA-mediated knockdown of TRPM4. When a higher concentration (10 mM) of BAPTA was included in the pipette solution, TICC activity was disrupted, suggesting that TRPM4 channels on the plasma membrane and IP3R on the SR are closely opposed but not physically coupled, and that endogenous Ca2+ buffer proteins play a critical role in maintaining TRPM4 channel activity in native cerebral artery smooth muscle cells.