Nicholas J. Sisco

Structural basis for KCNE3 modulation of potassium recycling in epithelia

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state. The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

Implications of Human Transient Receptor Potential Melastatin 8 (TRPM8) Channel Gating from Menthol Binding Studies of the Sensing Domain.

The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary cold sensor in humans. TRPM8 is gated by physiologically relevant cold temperatures and chemical ligands that induce cold sensations, such as the analgesic compound menthol. Characterization of TRPM8 ligand-gated channel activation will lead to a better understanding of the fundamental mechanisms that underlie TRPM8 function. Here, the direct binding of menthol to the isolated hTRPM8 sensing domain (transmembrane helices S1-S4) is investigated. These data are compared with two mutant sensing domain proteins, Y745H (S2 helix) and R842H (S4 helix), which have been previously identified in full length TRPM8 to be menthol insensitive. The data presented herein show that menthol specifically binds to the wild type, Y745H, and R842H TRPM8 sensing domain proteins. These results are the first to show that menthol directly binds to the TRPM8 sensing domain and indicates that Y745 and R842 residues, previously identified in functional studies as crucial to menthol sensitivity, do not affect menthol binding but instead alter coupling between the sensing domain and the pore domain.

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four‐carbon threose backbone in place of the five‐carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson–Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base‐pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A‐like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric “breathing” fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.

Statistical removal of background signals from high-throughput (1)H NMR line-broadening ligand-affinity screens.

NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction.