Evidence that the cold- and menthol-sensing functions of the human TRPM8 channel evolved separately.

Transient receptor potential melastatin 8 (TRPM8) is a temperature- and menthol-sensitive ion channel that contributes to diverse physiological roles, including cold sensing and pain perception. Clinical trials targeting TRPM8 have faced repeated setbacks predominantly due to the knowledge gap in unraveling the molecular underpinnings governing polymodal activation. A better understanding of the molecular foundations between the TRPM8 activation modes may aid the development of mode-specific, thermal-neutral therapies. Ancestral sequence reconstruction was used to explore the origins of TRPM8 activation modes. By resurrecting key TRPM8 nodes along the human evolutionary trajectory, we gained valuable insights into the trafficking, stability, and function of these ancestral forms. Notably, this approach unveiled the differential emergence of cold and menthol sensitivity over evolutionary time, providing a fresh perspective on complex polymodal behavior. These studies provide a paradigm for understanding polymodal behavior in TRPM8 and other proteins with the potential to enhance our understanding of sensory receptor biology and pave the way for innovative therapeutic interventions.

Recombinant SARS-CoV-2 envelope protein traffics to the trans-Golgi network following amphipol-mediated delivery into human cells.

The severe acute respiratory syndrome coronavirus 2 envelope protein (S2-E) is a conserved membrane protein that is important for coronavirus (CoV) assembly and budding. Here, we describe the recombinant expression and purification of S2-E in amphipol-class amphipathic polymer solutions, which solubilize and stabilize membrane proteins, but do not disrupt membranes. We found that amphipol delivery of S2-E to preformed planar bilayers results in spontaneous membrane integration and formation of viroporin cation channels. Amphipol delivery of the S2-E protein to human cells results in plasma membrane integration, followed by retrograde trafficking to the trans-Golgi network and accumulation in swollen perinuclear lysosomal-associated membrane protein 1-positive vesicles, likely lysosomes. CoV envelope proteins have previously been proposed to manipulate the luminal pH of the trans-Golgi network, which serves as an accumulation station for progeny CoV particles prior to cellular egress via lysosomes. Delivery of S2-E to cells will enable chemical biological approaches for future studies of severe acute respiratory syndrome coronavirus 2 pathogenesis and possibly even development of "Trojan horse" antiviral therapies. Finally, this work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.