RESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Canales Epiteliales de Sodio/metabolismo , Activación del Canal Iónico , Mecanotransducción Celular , Proteínas de la Membrana/metabolismo , Canales de Sodio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/genética , Relación Dosis-Respuesta a Droga , Canales Epiteliales de Sodio/química , Canales Epiteliales de Sodio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Microinyecciones , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Técnicas de Placa-Clamp , Conformación Proteica , Subunidades de Proteína , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/química , Canales de Sodio/efectos de los fármacos , Xenopus laevisRESUMEN
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.