The luminal K+ channel of the thick ascending limb of Henle's loop

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Abstract
In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10±12 cm3/s. The selectivity sequence für this channel was: K+=Rb+=NH 4 + =Cs+>Li+≫Na+=0; the conductance sequence was K+≫Li+≫Rb+=Cs+= NH 4 + =Na+=0. In excised patches Rb+, Cs+ and NH 4 + when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 μmol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities > 10±7 mol/l and by adenosine triphosphate ≥ 10±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by ≤ 0.2 units). The described channel differs in several properties from the K+ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K+ recycling in the TAL segment.

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