Jörg Grandl, Ph.D., PI
Pain and the sense of touch are mediated by ion channels that are activated by temperatures, chemicals, or mechanical stimuli. We use a combination of high-throughput screens and electrophysiology to understand functional mechanisms of these ion channels.
Mechanically-Activated Piezo Ion Channels
In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. However, many aspects of Piezo function remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. Our lab develops novel biophysical approaches to probe Piezo channel function:
For example, we expressed Piezo ion channels in cultured human kidney cells, and opened them by applying pressure to parts of the cell membrane inside a glass pipette. This causes a number of changes to the membrane, including to its curvature and tension, either of which could potentially open the Piezo channels. However, from images of the cell membrane inside the pipette we were able to calculate that tension is the activating stimulus. Further experiments unexpectedly revealed that the tension that is usually present in the cell membrane is sufficient to inactivate Piezo channels and prevent them from responding to a mechanical stimulus. This suggests that Piezo ion channels are inherently more sensitive to tension than previously realized, which could explain why different cell types appear to have different sensitivities to pressure (Lewis et al., eLife, 2015).
In another study, we were asking what specific parts (domains) of Piezo channels sense mechanical stimulation. To probe Piezos with sub-molecular resolution we developed a novel approach where we label specific domains within Piezos with magnetic nanoparticles and use an external magnetic field to generate a precise mechanical force that is highly localized within the channel protein. Simultaneously, we measure Piezo activation electrophysiologically. These experiments identified two distinct domains as being mechanically sensitive and involved in channel inactivation and activation (Wu et al., Nature Communications, 2016).
Next, we showed that Piezo ion channels function as pronounced frequency filters of repetitive mechanical stimulation, such as mechanical vibrations. Specifically, their transduction efficiencies depend on stimulus frequency, waveform, and duration, via a mechanism requiring intact channel inactivation. Our results show precisely what types of complex repetitive mechanical stimuli Piezo1 and Piezo2 ion channels are capable of transducing – and which ones they are not. For example, our results readily explain the previous finding that Piezo2 channels are involved in transducing the onset of mechanical vibrations, but not their continuation. (Lewis et al., Cell Reports, 2017).
Most recently, we showed that the C-terminal extracellular domain is mediating the specific time-course of inactivation between Piezo1 and Piezo2. We further identified a single charged lysine residue in the inner pore-helix of Piezo1 that determines the voltage-dependence of inactivation, and we showed by mutagenesis that the charge of this residue is sufficient to clamp inactivation kinetics independently of voltage. Altogether, our results suggest that the inner pore-helix undergoes voltage-dependent conformational changes that determine inactivation (Wu et al., Cell Reports, 2017).
Temperature-Activated Transient Receptor Potential (TRP) Ion Channels
The sense of warm and cold temperatures is mediated by transient receptor potential (TRP) ion channels that are activated by cold or hot temperatures at various thresholds. The molecular mechanism for the exceptional temperature sensitivity of TRP channels has been the focus of 20 years of research but is still poorly understood. We use random unbiased mutagenesis screens in combination with detailed functional characterization to uncover the mechanisms of temperature activation:
We performed an unbiased screen of 12,000 random mutant clones of the cold-sensitive ion channel mouse TRPA1. Using hot temperatures as a stimulus we identified several single-point mutations that invert temperature-directionality and make this ion channel sensitive to warm temperatures. All mutations are located within only one of 17 predicted ankyrin repeats, highlighting this domain as being important for the mechanism of temperature-activation. This finding suggests that both cold-activated and heat-activated TRP channels share one common mechanism of temperature activation (Jabba et al., Neuron, 2014).