The activation thresholds and inactivation kinetics of poking-evoked PIEZO1 and PIEZO2 currents are sensitive to subtle variations in mechanical stimulation parameters.

PIEZO1 and PIEZO2 are mechanically activated ion channels that confer mechanosensitivity to various cell types. PIEZO channels are commonly examined using the so-called poking technique, where currents are recorded in the whole-cell configuration of the patch-clamp technique, while the cell surface is mechanically stimulated with a small fire-polished patch pipette. Currently, there is no gold standard for mechanical stimulation, and therefore, stimulation protocols differ significantly between laboratories with regard to stimulation velocity, angle, and size of the stimulation probe. Here, we systematically examined the impact of variations in these three stimulation parameters on the outcomes of patch-clamp recordings of PIEZO1 and PIEZO2. We show that the inactivation kinetics of PIEZO1 and, to a lesser extent, of PIEZO2 change with the angle at which the probe that is used for mechanical stimulation is positioned and, even more prominently, with the size of its tip. Moreover, we found that the mechanical activation threshold of PIEZO2, but not PIEZO1, decreased with increasing stimulation speeds. Thus, our data show that two key outcome parameters of PIEZO-related patch-clamp studies are significantly affected by common variations in the mechanical stimulation protocols, which calls for caution when comparing data from different laboratories and highlights the need to establish a gold standard for mechanical stimulation to improve comparability and reproducibility of data obtained with the poking technique.

PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2.

PKA is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here, we examine the molecular mechanism underlying PKA-dependent modulation of the mechanically activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domain does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, though it remains unclear whether all or just some of these nine sites are required. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveal a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals.

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.

Role of TMEM100 in mechanically insensitive nociceptor un-silencing.

Mechanically silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors in mice. Moreover, we show that mice lacking TMEM100 do not develop secondary mechanical hypersensitivity-i.e., pain hypersensitivity that spreads beyond the site of inflammation-during knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce mechanical hypersensitivity in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary mechanical hypersensitivity during inflammation.

BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.