Biology subjects

Alvarez, O.

Publications and source records attributed to Alvarez, O..

5 recordsLinked to original sources

bioRxiv2025

Tuning the gate and the gear: The LRRC26 (γ1) subunit modulates intrinsic gating and voltage-sensor coupling of the BK channel

Association of auxiliary subunits ({beta}1-4 and {gamma}1-4) with the pore-forming subunit of the calcium- and voltage-activated potassium (BK) channel provides functional diversity. {gamma}1 promotes a significant leftward shift of the voltage activation curve, ensuring the adequate functioning of secretory glands, allowing the BK channel to release K+ at the cells resting Ca2+ concentration. Given its physiological importance, it is crucial to elucidate the mechanisms of {gamma}1 action. However, structural and functional studies have yielded conflicting conclusions regarding the modulation of BK channels by {gamma}1. Here, using macroscopic, single-channel, and gating current measurements, we demonstrate that at zero mV {gamma}1 increases 92-fold the equilibrium constant that defines the closed- open transition by destabilizing the channels closed configuration and enhancing the coupling between the voltage sensor and the pore domain, without affecting voltage-sensor activation. These results suggest that {gamma}1 not only causes an increase in the energetic coupling between the voltage sensors and the pore but mainly enhances the channel opening reaction. TeaserThe {gamma}1 subunit favors the BK channel pore opening by destabilizing its closed configuration.

biophysics↗

bioRxiv2023

Improved Estimation of Bird Abundance and Energy Consumption by Combining Point and Transect Observations

Point counts and transect surveys are both common methods for estimating bird abundance (by species, genus, or family) in a given environment. Sometimes both methods are used in the same study. Point counts reflect abundance directly, while transect observations of bird feeding instances reflect both the bird abundance and the mean energy consumption rate per bird. In this research we develop an improved estimator of bird abundance based on both sets of measurements, and derive error bars for these abundance estimates. The improved estimator is a linear combination of the two individual abundance estimates obtained from the two data types. The combination weights are obtained through a comparison of two separate energy estimates. The mathematical methods developed are applied to a dataset consisting of point counts and transect surveys of Ecuadorian frugivore bird species in six different types of environment (three elevations and two degrees of forestation). The improvement in accuracy achieved by this combined estimator is verified by simulations applied to bird family data. We also obtain 90% confidence intervals for the bird family relative abundance estimates, and verify the confidence intervals using simulations.

ecology↗

bioRxiv2022

A HUMANIZED FCRN TRANSGENIC MOUSE FOR PRECLINICAL PHARMACOKINETICS STUDIES

Monoclonal antibodies (mAbs) are one of the fastest-growing classes of drugs and have been approved to treat several diseases, including cancers and autoimmune disorders. Preclinical pharmacokinetics studies are performed to determine the therapeutically meaningful dosages and efficacy of candidate drugs. These studies are typically performed in non-human primates; however, using primates is costly and raises ethical considerations. As a result, rodent models that better mimic human-like pharmacokinetics have been generated and remain an area of active investigation. Pharmacokinetic characteristics of a candidate drug, such as half-life, are partly controlled by antibody binding to the human neonatal receptor hFCRN. Due to the abnormally high binding of human antibodies to mouse FCRN, traditional laboratory rodents do not accurately model the pharmacokinetics of human mAbs. In response, humanized rodents expressing hFCRN have been generated. However, these models generally use large inserts randomly integrated into the mouse genome. Here, we report the production and characterization of a CRISPR/Cas9-mediated hFCRN transgenic mouse termed SYNB-hFCRN. Using CRISPR/Cas9-assisted gene targeting, we prepared a strain with a simultaneous knockout of mFcrn and insertion of a hFCRN mini-gene under the control of the endogenous mouse promoter. These mice are healthy and express hFCRN in the appropriate tissues and immune cell subtypes. Pharmacokinetic evaluation of human IgG and adalimumab (Humira(R)) demonstrate hFCRN- mediated protection. These newly generated SYNB-hFCRN mice provide another valuable animal model for use in preclinical pharmacokinetics studies during early drug development. Materials availabilityCryopreserved germline cells from the strains are available from the MMRRC. Dr. Jarnagin will provide sequencing and other evidence supporting strain identity and the plasmids used for constructions.

immunology↗

bioRxiv2022

ROBUST AND EFFICIENT ACTIVE GENETICS GENE CONVERSION IN THE RAT AND MOUSE

The utility of Active Genetic (AG) gene conversion systems in rats and mice holds great promise for facilitating the production of complex strains harboring multiple humanizing genes. The practical application of such systems requires the identification of a robust, reusable, and highly efficient system. By characterizing twenty-eight different promoter and target site pairs we aimed to define the parameters needed to establish an efficient conversion system in male and female rats and mice. Using three specific meiosis prophase I active promoters to drive Cas9 expression. We studied several variables, including the number of Cas9 target sites, the distance between target sites, the cis versus trans configuration in linked pairs, and the effect of Cas9 copy number. In the rat, three of twelve tested configurations provided efficient AG gene conversion in the 22% - 67% range, and four others catalyzed AG in the 0.7-1% range. The rat Ddx4 (Vasa) promoter provides higher AG efficiency than the Sycp1 promoter. In mice, ten of sixteen tested configurations, using the Sycp1 and pSycp1 promoters, provided efficiency in the 0.3% - 3.2% range. In rats, Cas9 expression levels are remarkably well correlated with AG gene conversion efficiency. The rat cis rCyp3A1/rCyp3A2 locus was the most successful configuration, with gene conversion efficiencies of 0.7%-67%. This target site has a special property; the two Cas9 target sites are nearly perfectly homologous in the 100 bases around the gRNA target site. Our findings identify key parameters that improve AG efficiency, including the use of two Cas9 target sites, and efficient promoters that drive high levels of Cas9 expression that are correctly timed during gamete development. These findings also uncover the unexpected benefit of high homology at paired gRNA target sites to promote efficiency. We provide new data to guide future efforts to develop yet further improved AG systems.

genetics↗

bioRxiv2019

Calcium-driven regulation of voltage-sensing domains in BK channels

Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single -subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.

biophysics↗