Atrial myocytes are subjected to shear stress during the cardiac cycle under physiological or pathological conditions. The ionic currents regulated by shear stress remain poorly understood. We report the characteristics, molecular identity and activation mechanism of the shear stress-sensitive current (Ishear) in rat atrial myocytes. Atrial myocytes were enzymatically isolated from male Sprague-Dawley rats (230-300 g) and from wild-type and type 2 inositol 1,4,5,-trisphosphate receptor (IP3R) knock-out mice (C57/B6, 24-28 g) (anesthesia: pentobarbital sodium, 150 mg kg-1, i.p.). Shear stress of ~16 dyn cm−2 was applied to single myocytes using a pressurized microflow system, and the current was measured by whole-cell patch clamp. Values are means ± S.E.M., compared by student t test. In symmetrical CsCl solutions with minimal concentrations of internal EGTA, Ishear showed an outwardly rectifying current-voltage relationship (reversal at approximately −2.1±0.16 mV, n=62) and was well sustained. The current was conducted primarily (approximately 80%) by monovalent cations, but not Ca2+. It was suppressed by intracellular dialysis with 15 mM EGTA (p<0.0001 vs. 0.5 mM EGTA), selective inhibitors of transient receptor potential melastatin subfamily 4 (TRPM4) (p<0.01, 10 mM 9-phenanthrol vs. control; p<0.001, 100 mM 9-phenanthrol vs. control; p<0.01, 10 mM flufenamic acid vs. control), intracellular introduction of TRPM4 antibodies (p<0.001 vs. antibodies plus blocking peptides), or knock-down of TRPM4 expression (p<0.01 vs. wild-type), suggesting that TRPM4 carries most of this current. A notable reduction in Ishear occurred upon inhibition of Ca2+ release through the ryanodine receptors (p<0.05, 20 mM ryanodine vs. control; p<0.001, 50 mM ryanodine vs. control) or inositol 1,4,5-trisphosphate receptors (IP3Rs) (p<0.01, 2 mM 2-APB vs. control; p<0.05, 3 mM xestospongin C vs. control) and upon depletion of sarcoplasmic reticulum Ca2+ (p<0.01, 10 mM cyclopiazonic acid vs. control). In type 2 IP3R (IP3R2) knock-out atrial myocytes, Ishear was 10-20% of that in wild-type myocytes (p<0.01, wild-type vs. knock-out). Inhibition of protein kinase C, another protein activated by phospholipase C signaling, eliminated the sustained Ishear (p<0.01, control vs. 2 mM chelerythrine at 5-min shear), with no effect on initial Ishear (p>0.05, control vs. 2 mM chelerythrine at 30-s-min shear). Immunocytochemistry revealed that TRPM4 and IP3R2 were expressed at peripheral junctional sites with considerable co-localization. Our data suggest that shear stress activates TRPM4 current by triggering Ca2+ release from the IP3R2 in peripheral domains of atrial myocytes.