Stretch response of the mechano-gated channel TMEM63A in membrane patches and single cells.

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 ([Ca2+]i) and presented as (ΔF/F0 > 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 Gd3+. In whole cells stretched on flexible membranes, radial stretch increased the [Ca2+]i 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 [Ca2+]i increase in response to high stretch.NEW & NOTEWORTHY 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 [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel.

Voltage-gated ion channels (VGICs) orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. VGICs are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic voltage-gated sodium channel from Bacillus halodurans, to investigate mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel NaV1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis near the hinge diminished NaChBac mechanosensitivity, further supporting the proposed mechanism. Our results suggest that NaChBac is overall mechanosensitive due to the mechanosensitivity of a voltage-insensitive gating step associated with the pore opening. This mechanism may apply to eukaryotic VGICs, including NaV1.5.

Capsaicin as an amphipathic modulator of NaV1.5 mechanosensitivity.

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel that drives the electrical excitability of cardiac myocytes and contributes to slow waves of the human gastrointestinal smooth muscle cells. NaV1.5 is mechanosensitive: mechanical force modulates several facets of NaV1.5's voltage-gated function, and some NaV1.5 channelopathies are associated with abnormal NaV1.5 mechanosensitivity (MS). A class of membrane-active drugs, known as amphiphiles, therapeutically target NaV1.5's voltage-gated function and produce off-target effects including alteration of MS. Amphiphiles may provide a novel option for therapeutic modulation of NaV1.5's mechanosensitive operation. To more selectively target NaV1.5 MS, we searched for a membrane-partitioning amphipathic agent that would inhibit MS with minimal closed-state inhibition of voltage-gated currents. Among the amphiphiles tested, we selected capsaicin for further study. We used two methods to assess the effects of capsaicin on NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macro-patch and whole-cell Na+ current parameters with and without force. Capsaicin abolished the pressure- and shear-mediated peak current increase and acceleration; and the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of NaV1.5 MS makes capsaicin a promising candidate for therapeutic interventions targeting MS.

Sodium channels implement a molecular leaky integrator that detects action potentials and regulates neuronal firing.

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state. Through this mechanism, sodium channels effectively measure the frequency of action potentials and convert it into Na+ current availability. In turn, the Na+ current can control neuronal firing frequency in a negative feedback loop. Consequently, neurons become less sensitive to changes in excitatory input and maintain a lower firing rate. We present these ideas in the context of rat serotonergic raphe neurons, which fire spontaneously at low frequency and provide critical neuromodulation to many autonomous and cognitive brain functions.