|Budget Amount *help
¥9,400,000 (Direct Cost : ¥9,400,000)
Fiscal Year 1999 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 1998 : ¥5,400,000 (Direct Cost : ¥5,400,000)
To examine the extracellular Na sensitivity of a renal inwardly rectifying K channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K channel, ROMKI. When extracellular Na was removed, the whole-cell ROMKI currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMKI activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na from the pipette solution. However, macro-patch ROMKI currents recorded on the oocyte were significantly suppressed by Na removal. A blacker of Na/H antiporters, amiloride, largely inhibited the Na removal-induced suppression of whole-cell ROMKI currents. The pH-insensitive K80M mutant of ROMKI was much less sensitive to Na removal. Coexpression of ROMK1 with a Na/H antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMKI channels to extracellular Na in both the oocyt
es and HEK293 cells. Thus, it is concluded that the ROMKI channel is regulated indirectly by extracellular Na via intracellular pH changes.
Macula densa (MD) cells detect changes in tubular fluid composition through specific transport pathways and transmit signals which alter vascular resistance. Patch clamp studies were performed to define microscopic transport properties of these cells. Glomeruli were dissected from rabbit kidney and thick ascending limb removed to gain complete access to the macula densa. Patch clamp experiments in cell-attached (c/a) or inside- out (I/o) configurations were performed to directly observe ionic channels in MD cells. In c/a mode, we repeatedly observed a 20 pS channel with a linear I/Y which reversed near 0 mV. In I/o patches, the conductance was very similar, and the reversal potential was unaffected by replacing KCI with NaCl but outward currents disappeared upon bath replacement of all cautions with NMDG. Elimination of bath calcium (+ 1mM EGTA) abolished channel activity, and this was reversible upon readdition of calcium. Interestingly, this non-selective caution channel was found to be nifedipine-sensitive which suggests that it serve as a pathway for calcium.
In I/o patches, a large conductance anion channel was also identified with a linear current voltage relationship (mean conductance 383 pS). This channel reversed at 0 mV and displayed voltage inactivation for membrane potentials more positive than +30 mV. Channel activity was unaffected by removal of calcium and addition of EGTA but was blocked by gadolinium. The MD anion channel was also permeable to large anions including gluconate, aspartate, and, most interestingly, ATP. Single channel events in inside-out patches was found with 100 mM Na-ATP. In related studies, we used whole-cell conductance of PCI2 cells, placed close to the macula densa plaque, as a biosensor to monitor ATP release by MD cells. In response to an increase in bath NaCl from 25 to 150 mM, there was a significant release of ATP from MD cells. Interestingly, in parallel cell-attached experiments, maxi-CI channel activity was dependent upon the presence of bath [NaCl]. These results demonstrate, for the first time, that MD cells possess a maxi-CI channel which exhibits significant permeability to ATP and may release ATP in response to increases in [NaCl]. Less