Membrane Processes Mediating K+ Secretion

Weill Medical College Of Cornell Univ, New York NY

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Abstract

DESCRIPTION (provided by applicant): To preserve plasma K levels, the kidney excretes K in amounts that exactly match K intake. This is done at least in part by modulating the amount of K secreted into the urine through apical membrane K channels in the distal nephron. The movement of K through such channels will be studied both in renal tubular cells themselves and in Xenopus oocytes expressing the cloned renal K channel ROMK. The molecular determinants of ion conduction through the channels will be examined by exchanging parts of the C-terminus of ROMK with that of a related channel (IRK 1) which has different permeation properties. The role of the C-terminus will be further studied by assessing the ability of quaternary ammonium ions to enter the pore from the cytoplasmic side, and the ability of cysteine-modifying reagents to inhibit the function of channels with cysteines added to the C-terminus. The role of this part of the protein in the gating of the channel by internal pH and external K will also be explored by testing the ability of the channels to close when the cytoplasmic end is blocked by quaternary ammonium ions or polyamines. The basis for ion selectivity among K, Rb, TI and NH4 will be studied by measuring conductances with increasing concentrations of the various ions. Results will be compared with predictions made from discrete barrier models of ion permeation. The role of ROMK channels in mediating K secretion will be assessed by (1) measuring net K flux through the channels under various conditions of luminal K concentration and luminal membrane voltage (2) y measuring conducting channel densities under different physiological conditions and (3) applying this information to the development of a quantitative model of K transport by distal nephron segments. We will investigate the types of K channels (SK or ROMK channels, BK or Ca-activated K channels) expressed in the connecting tubule, a segment s previously demonstarted to take part in K secretion. Regulation of channels in this segment will be examined with particular focus on the role of dietary K (in vivo) and cAMP and glucagon-like peptide (in vitro). The possibility that hormones may promote the movement of channels between intracellular and plasma membrane compartments will be evaluated using immunocytochemistry of isolated renal tubules.