Competitive Interactions between PIRT, the Cold Sensing Ion Channel TRPM8, and PIP2 Suggest a Mechanism for Regulation.

TRPM8 is a member of the transient receptor potential ion channel family where it functions as a cold and pain sensor in humans and other higher organisms. Previous studies show that TRPM8 requires the signaling phosphoinositide lipid PIP2 to function. TRPM8 function is further regulated by other diverse mechanisms, including the small modulatory membrane protein PIRT (phosphoinositide regulator of TRP). Like TRPM8, PIRT also binds PIP2 and behavioral studies have shown that PIRT is required for normal TRPM8-mediated cold-sensing. To better understand the molecular mechanism of PIRT regulation of TRPM8, solution nuclear magnetic resonance (NMR) spectroscopy was used to assign the backbone resonances of full-length human PIRT and investigate the direct binding of PIRT to PIP2 and the human TRPM8 S1-S4 transmembrane domain. Microscale thermophoresis (MST) binding studies validate the NMR results and identify a competitive PIRT interaction between PIP2 and the TRPM8 S1-S4 domain. Computational PIP2 docking to a human TRPM8 comparative model was performed to help localize where PIRT may bind TRPM8. Taken together, our data suggest a mechanism where TRPM8, PIRT, and PIP2 form a regulatory complex and PIRT modulation of TRPM8 arises, at least in part, by regulating local concentrations of PIP2 accessible to TRPM8.

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABAA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABAA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Understanding thermosensitive transient receptor potential channels as versatile polymodal cellular sensors.

Transient receptor potential (TRP) ion channels are eukaryotic polymodal sensors that function as molecular cellular signal integrators. TRP family members sense and are modulated by a wide array of inputs, including temperature, pressure, pH, voltage, chemicals, lipids, and other proteins. These inputs induce signal transduction events mediated by nonselective cation passage through TRP channels. In this review, we focus on the thermosensitive TRP channels and highlight the emerging view that these channels play a variety of significant roles in physiology and pathophysiology in addition to sensory biology. We attempt to use this viewpoint as a framework to understand the complexity and controversy of TRP channel modulation and ultimately suggest that the complex functional behavior arises inherently because this class of protein is exquisitely sensitive to many diverse and distinct signal inputs. To illustrate this idea, we primarily focus on TRP channel thermosensing. We also offer a structural, biochemical, biophysical, and computational perspective that may help to bring more coherence and consensus in understanding the function of this important class of proteins.