G protein-activated inwardly rectifying K(+) channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1 and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by PKC activation (PMA). The mechanosensitivity of GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1(K207-L245)) and single point mutant (GIRK4(I229L)), in which PIP(2) binding domain or residue was replaced by corresponding region of IRK1 to strengthen the channel-PIP(2) interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP(2) binding domain (L222I/R213Q). Overexpression of Gbetagamma, which is known to strengthen the channel-PIP(2) interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-delta tagged with GFP, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP(2) hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K(+) channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP(2) interaction, with PKC playing an important role in modulating the interaction probably through PIP(2) hydrolysis.