Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae.

In this study, amiodarone, at very low concentrations, produced a clear efflux of K(+). Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K(+) efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca(2+) and H(+), the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca(2+) concentration, as well as the decreased internal pH. The Deltatok1 and Deltanha1 mutations resulted in a smaller effect of amiodarone, and Deltatrk1 and Deltatrk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K(+), through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.

The yeast potassium transporter TRK2 is able to substitute for TRK1 in its biological function under low K and low pH conditions.

In S. cerevisiae, K+ transport relies principally on two structurally related membrane proteins, known as Trk1p and Trk2p. Direct involvement in cation movements has been demonstrated for Trk1p, which is a high-affinity K+ transporter. Initially described as a low-affinity K+ transporter, Trk2p seems to play a minor role in K+ transport, since its activity is only apparent under very specific conditions, such as in a Deltasin3 background. Here we show that growth of a Deltatrk1Deltasin3 double mutant, under K+-limiting conditions or at low pH, is Trk2p-dependent, and by Northern blot analysis we demonstrate that deletion of SIN3 results in transcriptional derepression of TRK2. In addition, we show that heterologous overexpression of TRK2 with the inducible GAL1 promoter bypasses Sin3p repression in a Deltatrk1Deltatrk2 double mutant and fully restores growth under non-permissive conditions. Furthermore, kinetic experiments in a Deltatrk1Deltasin3 double mutant revealed a K+ transporter with an apparent high affinity and a moderate capacity. Taken together, these results indicate that TRK2 encodes a functional K+ transporter that, under our experimental conditions, displays distinctive kinetic characteristics.

The SUMO ubiquitin-protein isopeptide ligase family member Miz1/PIASxbeta /Siz2 is a transcriptional cofactor for TFII-I.

We have shown previously that a TFII-I-related protein, hMusTRD1/BEN, represses transcriptional activity of TFII-I. The repression by hMusTRD1/BEN was hypothesized to occur via a two-step competition mechanism involving a cytoplasmic shuttling factor and a nuclear cofactor required for transcriptional activation of TFII-I. Employing a two-hybrid approach with both yeast genomic and mouse cDNA libraries in parallel, we have identified the RING-like zinc finger containing Miz1/PIASxbeta/Siz2, which is a ubiquitin-protein isopeptide ligase in the SUMO pathway, as the potential nuclear cofactor that interacts with both TFII-I and hMusTRD1/BEN. Our conclusion is based on the following observations. First, the interactions are biochemically confirmed in mammalian cells where Miz1/mPIASxbeta interacts with both TFII-I and hMusTRD1/BEN when these proteins are ectopically co-expressed. Second, co-expression of a nuclear localization signal-deficient mutant of Miz1/mPIASxbeta with wild type TFII-I fails to alter the subcellular localization of the former. Finally, ectopically expressed Miz1/mPIASxbeta augments the transcriptional activity of TFII-I and relieves the repression exerted by a mutant hMusTRD1/BEN that co-localized with TFII-I in the nucleus.

Bases moleculares de la hipertension arterial inducida por cadmio / Molecular basis of the cadmium induced by arterial hypertension

Con el fin de esclarecer los mecanismos bioquímicos involucrados en el establecimiento de la enfermedad hipertensiva inducida por el cadmio, en este trabajo se estudió el efecto del metal sobre diversas funciones mitocondriales, a saber: transporte de calcio y producción de energía. La presencia de cadmio en el medio inhibe ambas funciones debido a la unión de este metal a los grupos sulfhidrilo presentes en las proteínas de las cuales dependen estos procesos. En base a estos resultados es posible proponer un esquema en el cual el cadmio produce de manera directa un efecto de vasoconstricción renal el cual a su vez explica otros mecanismos que aparecen durante la enfermedad hipertensiva