Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade.

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.

Unidad de dolor agudo posoperatorio. Casuística del Hospital de Clínicas / Post-surgery acute pain Unit. Hospital de Clínicas

El dolor agudo posoperatorio tiene una alta prevalencia y las unidades de dolor agudo han demostrado ser la mejor forma de tratarlo. Se presenta la casuística de la primera unidad del país, que funciona en el Departamento de Anestesiología del Hospital de Clínicas. Se analizan en forma prospectiva 617 pacientes quirúrgicos, evaluando la incidencia e intensidad del dolor mediante la escala numérica análoga (ENA). De acuerdo a la intensidad del dolor se aplicaron protocolos farmacológicos, que se inician desde el intraoperatorio: dolor leve: antiinflamatorios no esteroideos (AINEs); dolor moderado: AINEs más opiode débil; dolor severo: opiode fuerte. Se previó la colocación de catéter peridural lumbar o torácico en la cirugía abdominal mayor o torácica. Al ingreso a sala de recuperación posanestésica (SRPA) hubo una incidencia de dolor de 44 por ciento: 11 por ciento dolor leve, 15 por ciento moderado y 18 por ciento severo. Luego del ajuste individualizado de la analgesia, se registró una disminución significativa de la incidencia (21 por ciento) y severidad del dolor (20,7 por ciento dolor leve y 0,3 por ciento moderado). A las 24 horas en sala de internación se observa un aumento de la incidencia del dolor en sus tres categorías (leve 27 por ciento, moderado 11 por ciento y severo 3 por ciento). 555 pacientes (90 por ciento) fueron dados de alta a las 24 horas, 41 (7 por ciento) a las 48 horas y 21 (3 por ciento) a las 72 horas de la Unidad de Dolor Agudo (UDA). La UDA permitió dar una mejor solución al dolor posoperatorio, uno de los aspectos que más afectan la calidad de la recuperación.

The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots.

When chilling-sensitive plants are chilled, root hydraulic conductance (L(o)) declines precipitously; L(o) also declines in chilling-tolerant plants, but it subsequently recovers, whereas in chilling-sensitive plants it does not. As a result, the chilling-sensitive plants dry out and may die. Using a chilling-sensitive and a chilling-tolerant maize genotype we investigated the effect of chilling on L(o), and its relationship to osmotic water permeability of isolated root cortex protoplasts, aquaporin gene expression, aquaporin abundance, and aquaporin phosphorylation, hydrogen peroxide (H(2)O(2)) accumulation in the roots and electrolyte leakage from the roots. Because chilling can cause H(2)O(2) accumulation we also determined the effects of a short H(2)O(2) treatment of the roots and examined the same parameters. We conclude from these studies that the recovery of L(o) during chilling in the chilling-tolerant genotype is made possible by avoiding or repairing membrane damage and by a greater abundance and/or activity of aquaporins. The same changes in aquaporins take place in the chilling-sensitive genotype, but we postulate that membrane damage prevents the L(o) recovery. It appears that the aquaporin response is necessary but not sufficient to respond to chilling injury. The plant must also be able to avoid the oxidative damage that accompanies chilling.