Master of Science / Department of Kinesiology / Steven W. Copp / Mechanical signals within contracting skeletal muscles contribute to the generation of the exercise pressor reflex; an important autonomic and cardiovascular control mechanism. In decerebrate rats, GsMTx4, a mechanically–activated channel inhibitor that is partially selective for piezo channels, was found recently to reduce the pressor response during static hindlimb muscle stretch; a maneuver used to investigate the mechanical component of the exercise pressor reflex (i.e., the mechanoreflex). However, the effect was found only during the very initial phase of the stretch when muscle length was changing which may have reflected the inhibition of rapidly-deactivating piezo 2 channels and the fact that different mechanically-activated channels with slower deactivation kinetics evoked the pressor response during the static phase of the maneuver. We tested the hypothesis that in decerebrate, unanesthetized rats, GsMTx4 would reduce the pressor response throughout the duration of a 30 second, 1 Hz dynamic hindlimb muscle stretch protocol. We found that the injection of 10 µg of GsMTx4 into the arterial supply of a hindlimb reduced the peak pressor response (control: 15±4, GsMTx4: 5±2 mmHg, p<0.05, n=8) and the pressor response at multiple time points throughout the duration of the stretch. GsMTx4, however, had no effect on the pressor response to the hindlimb arterial injection of lactic acid. Moreover, the injection of GsMTx4 into the jugular vein (a systemic control, n=5) or the injection of saline into the hindlimb arterial supply (a vehicle control, n=4) had no effect on the pressor response during dynamic stretch. We conclude that GsMTx4 reduced the pressor response throughout the duration of a 1 Hz dynamic stretch protocol which may have reflected the inhibition of piezo 2 channels throughout the dynamic stretch maneuver.

Organisms are constantly subjected to mechanical stimuli originating from their environment or internal activities. To sense and properly handle such mechanical cues, the conversion of mechanical signals into electrochemical signals occurs continuously within the organisms. Mechanosensitive ion channels (MSCs) are pivotal participants in this process, being ready to open to allow the movement of ions across cellular membranes in response to mechanical stimulation. Piezo1 and Piezo2 mechanosensitive cation channels are evolutionarily conserved MSCs, taking part in various physiological processes, including bone formation and touch sensation. Mutations in Piezo1 or Piezo2 lead to pathological conditions and overexpression of the channels is associated with several diseases. Piezo modulators can be used not only as useful tools for targeted interventions of such pathological outcomes but also as important contributors to the characterization and study of Piezo channels. This dissertation is a part of such endeavors to identify Piezo modulators. Proteinogenic sulfur-containing amino acids (PSCAAs), which is a term to call methionine (Met) and cysteine (Cys) collectively, and propofol were chosen as candidates for modulating Piezo channel activities, given their potential to modify membranes as gating of Piezo channels is known to be greatly influenced by mechanical properties of cellular membranes.The effects of PSCAAs and propofol on Piezo channels were evaluated through electrophysiological recording and calcium imaging, which were performed using Pieoz1-deficient (P1KO) HEK293T cells transfected with either human Piezo1 or human Piezo2 and HEK293T cells that stably overexpress Piezo1, respectively. It was observed that perfusion of 100 µM Met on Piezo1-transfected P1KO HEK293T enhanced Piezo1 currents and, interestingly, delayed Piezo1 inactivation, in the whole-cell and outside-out configurations. However, 100 µM D-Met was incapable of inducing calcium influx into the Piezo1-overexpressing cell line in calcium imaging. Double-mutant Piezo1, which is unable to inactivate due to a substitution of arginine for Met (M2225R) and a substitution of lysine for arginine (R2456K), was unaffected by 100 µM D-Met in the outside-out configuration. These results indicate that Met augments Piezo1 currents by slowing down Piezo1 inactivation instead of contributing to activation of more Piezo1 channels. Piezo2 currents, on the other hand, were not altered by 100 µM D-Met in the cell-attached and whole-cell configurations. 100 µM Cys induced responses similar to those of Met, increasing Piezo1 currents while delaying inactivation. 50 µM propofol inhibited Piezo1 currents in outside-out, whole-cell, and cell-attached recording and counteracted Yoda1-induced calcium influx into Piezo1-overexpressing HEK293T cells. Piezo2 currents were also suppressed by 50 µM propofol. This study reveals that PSCAAs enhance Piezo1 current while delaying its inactivation and propofol inhibits both Piezo1 and Piezo2 channels. These findings offer potential strategies for targeted therapeutic modulation of Piezo channels, advancing the treatment options for Piezo-related conditions and expanding our understanding of mechanosensory signal regulation.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24637号 / 工博第5143号 / 新制||工||1982(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM