Biology subjects

Hernandez, C. Q.

Publications and source records attributed to Hernandez, C. Q..

4 recordsLinked to original sources

bioRxiv2025

Insights into the structure and modulation of human TWIK-2

The Tandem of pore domain in a Weak Inward Rectifying K+ channel 2 (TWIK-2; KCNK6) is a member of the Two-Pore Domain K+ (K2P) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. The structure and regulatory mechanisms of TWIK-2 remain largely unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure of human TWIK-2 at [~]3.7 [A] and highlight its conserved and unique features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing structure and sequence-guided site-directed mutagenesis and provide insights into the possible mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising new anti-inflammatory therapeutics.

biophysics↗

bioRxiv2024

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

neuroscience↗

bioRxiv2020

Membrane Orientation and Oligomerization of the Melanocortin Receptor Accessory Protein 2

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G-protein coupled receptors (GPCR) that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates GPCRs and will aid in elucidating the pathways involved in metabolic dysfunction.

biochemistry↗

bioRxiv2018

Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulate KCNQ3 K+ Channels Via Multiple Sites Of Action

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the function of many ion channels, including M-type (KCNQ1-5, Kv7) K+ channels; however the molecular mechanisms involved in this regulation remain unclear. To identify the sites of action on KCNQ3 channels, we used as our baseline the A315T pore mutant (KCNQ3T) that increases channel currents without modifying the apparent affinity of PIP2 and performed extensive mutagenesis in regions that have been suggested to be involved in PIP2 interactions among the KCNQ family. Using the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe for apparent affinity of the channels, we found that PIP2 modulates KCNQ channel function through four different domains. 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix (S6Jx), 3) the S2-S3 linker and 4) the S4-S5 linker. We found that PIP2 interactions within these domains were not coupled to the voltage dependence of activation. Extensive homology modeling and docking simulations between the wild-type or mutant KCNQ3 channels and PIP2, correlated with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

neuroscience↗