ИСТИНА

Living cells express a number of mechano-dependent molecules that transduce mechanical forces and trigger a variety of intracellular signaling events. One of the major players is mechanosensitive ion channels, whose activation and inactivation is regulated by the mechanical status of the cell. Calcium influx via non-selective Ca2+-permeable channels activated by membrane stretch (stretch-activated channels, SACs) is thought to have an valuable impact on intracellular signaling and calcium-dependent coupling of cellular reactions. Previously we have identified typical SACs in 3T3B-SV40 mouse transformed fibroblasts with unitary conductance of 24.4±1.4 ps (mean ± S.E.M). Importantly, we showed that calcium influx via SACs triggered the activation of calcium-activated potassium channels (Chubinskiy-Nadezhdin et al., 2014). The goal of the present study was to identify the main molecular candidate for the role of SAC in 3T3B-SV40 cells using a combination of pharmacological approach and molecular biology techniques. The proposed candidates were proteins from TRP superfamily (TRPC1, C6, V2, V4 and M7), ASIC/ENaCs and Piezo1/2. The pharmacological blockers of putative mechanosensitive channels were added to the patch pipette in cell-attached single-channel patch-clamp experiments. The following inhibitors were tested: blocker of TRPC1 and C6 La3+ (100 µM), blocker of TRPV subfamily Ruthenium Red (10 µM), blocker of TRPC1/6 and activator of TRPV2/4 2-APB (100 µM) and amiloride (200 µM , blocker of ASIC/ENaC superfamily and TRPC6). All of selected compounds had no effect on stretch-induced activation of SACs and single channel conductance in the plasma membrane. The unique fingerprint of TRPM7 is the dramatic increase of the unitary conductance in the absence of Mg2+ ions (Macianskicne et al., 2012), The variation of Mg2+ concentration had no effect on the unitary conductance of SACs, thus TRPM7 was excluded from the possible correlates. In sum, the experiments with the use of pharmacological blockers allow us to identify Piezo1/2 as the main candidates on the role of SACs in transformed fibroblasts. The RT-PCR detected the presence of PIEZO1 mRNA but not PIEZO2 in cells. Immunofluorescent staining with specific acti-Piezo1 antibodies confirmed the expression of Piezo1 in the cell membrane. Importantly, a predominant localization of Piezo1 with in lamellopodia regions was detected by double immunofluorescent staining of Piezo1 and F-actin. No significant co-localization of Piezo1 with lipid raft marker GM1 was observed, and the channel activity was not affected by lipid raft disruption with methyl-beta-cyclodextrin. The role of Piezo1 in the signaling processes (e.g. cell migration and calcium signaling) in 3T3B-SV40 mouse transformed fibroblasts is planned to be studied using selective siRNA gene knockdown. The work was supported by RFBR grants 15-04-00905 and 15-04-02950.