Journal articles

The effects of the temperature-sensitive, immortalizing Simian Virus 40 T antigen, tsA58, on whole-cell potassium conductances were assessed in renal glomerular mesangial cells from H-2Kb-tsA58 transgenic mice [1]. MTT cell viability assay data indicated that in permissive (33 degrees C, 50 U ml-1 gamma-interferon, IFN+) and non-permissive (37 degrees C, without gamma-interferon, IFN-) culture conditions the oncogene was active and inactive respectively.

1. Depolarization of mesangial cells has been shown to occur following an outward movement of chloride ions from the cell. We have shown previously that mesangial cells from the H-2Kb-tsA58 transgenic mouse possess a significant whole-cell chloride conductance and consequently are a suitable preparation for the study of potential chloride channel inhibitors. 2.

The effects of duration in culture were assessed in mesangial cells from the H-2Kb-tsA58 transgenic mouse using the whole-cell configuration of the patch clamp technique. The whole-cell potassium conductance remained constant in cells from passages 8-17 while the chloride conductance was found to decrease in cells after passage 15. This reduction of the chloride conductance, indicating cell dedifferentiation, was the result of complete loss of the calcium-activated component of the conductance and loss of part of the calcium-insensitive component.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1.

Anion transport in human multidrug-resistant large cell lung tumour cells (COR-L23/R) which overexpress the multidrug-resistance-associated protein (MRP) has been compared with that in cells of the parent line (COR-L23/P). Whole-cell patch-clamp recordings reveal variability between individual cells in basal anion conductance and in anion conductance increases following exposure to hypotonic media. The increase of stimulated over basal conductance is significantly larger for resistant cells than for parent cells.

1. The patch-clamp technique, both cell attached and inside-out patches, was used to examine the effects of lysylbradykinin (LBK) and A23187 on ion channels in cultured Colony 29 epithelial cells derived from a human adenocarcinoma. 2. LBK and A23187 applied directly to the intact cell stimulated the opening of a number of types of ion channel including Ca(2+)-activated K+ channels. 3. By use of inside-out patches, anion channels could be stimulated to open by application of protein kinase A and ATP to the cytosolic surface.

The chloride conductance of conditionally immortalized mesangial cells isolated from the H-2Kb-tsA58 transgenic mouse was studied in cells grown in permissive and non-permissive culture conditions. No differences were found in the magnitude of the chloride conductance in 140 mM tetramethyl ammonium chloride between cells grown in permissive and non-permissive culture conditions (1.08 +/- 0.05 nS and 1.02 +/- 0.05 nS).

The acid-sensing ion channel (ASIC) 1a is known to assemble as a homotrimer. Here, we used atomic force microscopy to image ASIC1a, integrated into lipid bilayers, at pH 7.0 and pH 6.0. The triangular appearance of the channel was clearly visible. A height distribution for the channels at pH 7.0 had two peaks, at 2 and 4 nm, likely representing the intracellular and extracellular domains, respectively. At pH 6.0 the 2-nm peak remained, but the higher peak shifted to 6 nm. Hence, the extracellular domain of the channel becomes 'taller' after acidification.

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel.