Biology subjects

Gaete, P. S.

Publications and source records attributed to Gaete, P. S..

3 recordsLinked to original sources

bioRxiv2024

Impaired S-nitrosylation of Cx43 prevents arrhythmogenicity and myocardial injury upon cardiac stress in Duchenne Muscular Dystrophy

Connexin-43 (Cx43) plays a critical role in the propagation of action potentials and cardiac contractility. In healthy cardiomyocytes, Cx43 is mainly located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Using a mouse model of Duchenne muscular dystrophy (DMD), we previously demonstrated that Cx43 localizes to the lateral side of dystrophic cardiomyocytes, forming undocked hemichannels. {beta}-adrenergic signaling-induced cardiac stress promotes S-nitrosylation and the opening of undocked Cx43 hemichannels leading to disrupted cardiac membrane excitability and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we generated knockin DMDmdx mice with reduced levels of S-nitrosylated Cx43, by replacing cysteine 271 with a serine in one Cx43 of the unique site for S-nitrosylation of Cx43 (DMDmdx:C271S+/-). Immunofluorescence analysis revealed that cardiac Cx43 lateralization in DMDmdx:C271S+/- mice was similar to DMDmdx mice, indicating that the genetic modification did not prevent Cx43 remodeling. Upon isoproterenol treatment, DMDmdx mice displayed a higher incidence of arrhythmogenic events when compared to DMDmdx:C271S+/- mice, which more closely resemble wild-type mice. Optical mapping imaging in isolated hearts showed that DMDmdx mice displayed aberrant Ca2+ signaling and prolonged action potentials, which is restored in DMDmdx:C271S+/- mice. Isoproterenol treatment evoked severe myocardial injury in DMDmdx mice, which was significantly attenuated in DMDmdx:C271S+/- mice. Notably, DMDmdx mice treated with Gap19, a Cx43 hemichannel blocker, exhibited cardioprotection against myocardial injury. We concluded that S-nitrosylation of Cx43 proteins is a fundamental NO-mediated mechanism involved in arrhythmias and myocardial injury in DMDmdx, occurring through the opening of hemichannels following {beta}-adrenergic stress.

physiology↗

bioRxiv2024

Large-pore connexin hemichannels function as molecule transporters independently of ion conduction

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore -- a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

biophysics↗

bioRxiv2020

A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels.

Channels that are permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, mechanisms of molecular permeation and selectivity of these channels remain mostly unexplored due to the lack of quantitative methodologies. To address this need, we developed a novel two-electrode voltage clamp (TEVC)/dye uptake assay to examine the kinetics of molecular permeation of channels formed by human connexins (hCx), and the calcium homeostasis modulator (hCALHM1). We expressed hCx26, hCx30, and hCALHM1 individually in Xenopus laevis oocytes. To quantify the uptake of small molecular dyes through these channels, we developed a protocol that renders oocytes translucent - thereby amenable to optical detection techniques - without affecting the functional properties of the expressed channels. To control membrane potential and to determine functional channel expression accurately, dye uptake was evaluated in conjunction with TEVC. Using this methodology, we found that: (1) CALHM1 and Cx30 hemichannels display saturable transport of molecules that could be described by Michaelis-Menten kinetics, with apparent KM and Vmax; (2) Kinetic parameters for molecular transport through CALHM1 are sensitive to voltage and extracellular calcium; (3) Significant transport of molecules occurs through CALHM1 when there are little or no ionic currents through the channels; (4) Cx mutations in the N-terminal region significantly affect kinetics of transport and permselectivity. Our results reveal that molecular flux through these channels has a rate-limiting step, that the kinetic parameters of molecular transport are sensitive to modulators of channel gating and that molecular transport and ionic currents can be differentially affected. Our methodology allows the analysis of how human mutations causing diseases affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, associated with molecular transport in large-pore channels.

biophysics↗