Global twisting motion of single molecular KcsA potassium channel upon gating

Ion channels are signal transduction molecules that switch ion permeation pathways on and off (gating). Crystal structures of several kinds of potassium channels have revealed open and closed conformations, which provide static pictures of gating status. Here we studied KcsA potassium channels undergoing conformational changes at the single-molecule level. A KcsA channel with a gold nanocrystal attached was irradiated by white X-rays and motions of the diffraction spot from the nanocrystal were tracked in real time. Upon gating, the KcsA channels twisted around the axis of the pore. These conformational changes were prevented by an open-channel blocker, tetrabuthylammonium. Random clockwise and counterclockwise twisting in the range of several tens of degrees originated in the transmembrane domain and was transmitted to the cytoplasmic domain. This coupling suggests a mechanical interplay between the transmembrane and cytoplasmic domains.     

Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.     

Factors responsible for oscillations of membrane potential recorded with tight-seal-patch electrodes in mouse fibroblasts

Summary In giant fibroblastic L cells, penetration of a conventional microelectrode brought about marked decreases in the membrane potential and input resistance measured with a patch electrode under tight-seal whole-cell configuration, and repeated hyperpolarizations were often observed upon penetration. Therefore, the question arose whether such leakage artifact is a causal factor for generation of the membrane potential oscillation even in giant L cells. During whole-cell recordings, however, regular potential oscillations were observed in the cells that had not been impaled with a conventional microelectrode, as far as the Ca²⁺ buffer was not strong in the pipette solution. Oscillatory changes in the intracellular potential were detected by extracellular recordings with a tight-seal patch electrode in the cell-attached configuration. Thus, the potential oscillation occurs even in the absence of penetration-induced leakage or without rupture of the patch membrane. Withdrawal of a micropipette from one cell was often found to induce marked cell damage and elicit oscillatory hyperpolarizations in a neighboring cell with a certain time lag. The longer the distance between the injured and recorded cells, the greater was the time lag. Application of the cell lysate on the cell surface also gave rise to oscillatory hyperpolarizations. After repeated applications of the lysate, the membrane became unresponsive (desensitized), suggesting the involvement of receptors for the lysate factor. The lysates of different cell species (mouse lymphoma L5178Y cells or human epithelial Intestine 407 cells) produced similar effects. The effective component was heat stable and distinct from ATP. Lysate-induced hyperpolarizations were inhibited by deprivation of extracellular Ca²⁺ and by application of a Ca²⁺ channel blocker (nifedipine) or a K⁺ channel blocker (quinine) in the same manner as spontaneous oscillatory hyperpolarizations. It is concluded that the mouse fibroblast exhibits membrane potential oscillations, when the cell was activated, presumably via receptor systems, by some diffusible factors released from damaged cells.