|
ИСТИНА |
Войти в систему Регистрация |
ИСТИНА ЦЭМИ РАН |
||
Mechanosensitive calcium-permeable ion channels in transformed fibroblasts: An evidence for Piezo1статья Тезисы
- Авторы: Chubinskiy-Nadezhdin V., Vassilieva I., Vasileva V., Negulyaev Y., Morachevskaya E.
- Журнал: Proceedings of The Physiological Society
- Том: 37
- Год издания: 2016
- Первая страница: PCA120
- Последняя страница: PCA120
- Аннотация: 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. Importantly, we showed that calcium influx via SACs triggered the activation of calcium-sensitive 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/C6 lanthanium (100 µM), blocker of TRPV subfamily Ruthenium Red (10 µM), blocker of TRPC1/6 and activator of TRPV2/4 2-APB (100 µM), amiloride (200 µM, blocker of ASIC/ENaC and TRPC6). We showed that all tested compounds had no effect on stretch-induced activation of SACs and single channel conductance in the plasma membrane. It is known, that the unique fingerprint of TRPM7 is the dramatic increase of the unitary conductance in the absence of Mg2+ ions (Macianskiene et al., 2012). 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 suggest Piezo1/2 as the main candidates on the role of SACs in transformed fibroblasts. RT-PCR analysis detected the presence of PIEZO1 mRNA but not PIEZO2 mRNA in cells. Immunofluorescent staining with the specific anti-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 staining of Piezo1 and F-actin. It is worth noting that actin disassembly by cytochalasin D did not prevent coupling of mechano-gated and calcium-activated channels in course of mechanotranduction. The role of Piezo1 in the signaling processes (e.g. cell migration, motility and calcium signaling) in 3T3B-SV40 transformed fibroblasts is planned to be studied using selective siRNA gene knockdown.
- Добавил в систему: Негуляев Юрий Алексеевич