Niloy Sayeman Islam, Strege Peter R, Hannan Elizabeth C, Cowan Luke M, Linsenmeier Fabian, Friedrich Oliver, Farrugia Gianrico, Beyder Arthur
Enteric Neuroscience Program (ENSP), Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, United States.
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, United States.
Am J Physiol Cell Physiol. 2024 Feb 1;326(2):C622-C631. doi: 10.1152/ajpcell.00583.2023. Epub 2024 Jan 8.
The recently discovered ion channel TMEM63A has biophysical features distinctive for mechano-gated cation channels, activating at high pressures with slow kinetics while not inactivating. However, some biophysical properties are less clear, including no information on its function in whole cells. The aim of this study is to expand the TMEM63A biophysical characterization and examine the function in whole cells. Piezo1-knockout HEK293T cells were cotransfected with human TMEM63A and green fluorescent protein (GFP), and macroscopic currents in cell-attached patches were recorded by high-speed pressure clamp at holding voltages from -120 to -20 mV with 0-100 mmHg patch suction for 1 s. HEK293 cells cotransfected with TMEM63A and GCaMP5 were seeded onto polydimethylsiloxane (PDMS) membrane, and the response to 3-12 s of 1%-15% whole cell isotropic (equi-biaxial) stretch induced by an IsoStretcher was measured by the change in intracellular calcium ([Ca]) and presented as (ΔF/F > 1). Increasing patch pressures activated TMEM63A currents with accelerating activation kinetics and current amplitudes that were pressure dependent but voltage independent. TMEM63A currents were plateaued within 2 s, recovered quickly, and were sensitive to Gd. In whole cells stretched on flexible membranes, radial stretch increased the [Ca] responses in a larger proportion of cells cotransfected with TMEM63A and GCaMP5 than GCaMP5-only controls. TMEM63A currents are force activated and voltage insensitive, have a high threshold for pressure activation with slow activation and deactivation, and lack inactivation over 5 s. TMEM63A has the net polarity and kinetics that would depolarize plasma membranes and increase inward currents, contributing to a sustained [Ca] increase in response to high stretch. TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca] responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca] increase.
最近发现的离子通道TMEM63A具有机械门控阳离子通道独特的生物物理特性，在高压下激活，动力学缓慢且不会失活。然而，一些生物物理特性尚不清楚，包括其在全细胞中的功能信息缺失。本研究的目的是扩展TMEM63A的生物物理特性表征并研究其在全细胞中的功能。将Piezo1基因敲除的HEK293T细胞与人TMEM63A和绿色荧光蛋白（GFP）共转染，在细胞贴附式膜片钳中，通过高速压力钳在-120至-20 mV的钳制电压下，以0 - 100 mmHg的膜片负压抽吸1 s记录宏观电流。将共转染TMEM63A和GCaMP5的HEK293细胞接种到聚二甲基硅氧烷（PDMS）膜上，通过细胞内钙（[Ca]）的变化测量IsoStretcher诱导的1% - 15%全细胞各向同性（等双轴）拉伸3 - 12 s的反应，并表示为（ΔF/F > 1）。增加膜片压力可激活TMEM63A电流，激活动力学加速，电流幅度与压力相关但与电压无关。TMEM63A电流在2 s内达到平台期，快速恢复，且对钆敏感。在柔性膜上拉伸的全细胞中，与仅转染GCaMP5的对照相比，径向拉伸在更大比例的共转染TMEM63A和GCaMP5的细胞中增加了[Ca]反应。TMEM63A电流由力激活且对电压不敏感，压力激活阈值高，激活和失活缓慢，且在5 s内不失活。TMEM63A具有使质膜去极化并增加内向电流的净极性和动力学，有助于在高拉伸时持续增加[Ca]。TMEM63A具有机械门控阳离子通道独特的生物物理特性，但一些特性尚不清楚，包括在全细胞中的功能信息缺失。我们报告细胞膜片钳中压力依赖性但电压非依赖性的TMEM63A电流与细胞大小相关。此外，柔性膜上全细胞的径向拉伸在转染TMEM63A的细胞中更能增加[Ca]反应。响应高拉伸的内向TMEM63A电流可使质膜去极化并有助于持续增加[Ca]。
