Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

1. We characterized chloride currents in freshly isolated rabbit osteoclasts using whole‐cell and single channel patch‐clamp recording configurations. Depolarization activated an outwardly rectifying current in 40‐50% of cells, distinct from the inwardly rectifying K+ current we have previously reported in osteoclasts. 2. The outwardly rectifying current persisted under conditions where all K+ currents were blocked. Furthermore, the outward current was reversibly inhibited by Cl‐ transport blockers 4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid (SITS); 4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (DIDS); 4,4'‐dinitrostilbene‐2,2'‐disulphonic acid (DNDS); and niflumic acid. The blocked current had a reversal potential close to the predicted chloride equilibrium potential and was dependent on the chloride concentration gradient. 3. In those osteoclasts in which outwardly rectifying current was not initially apparent, exposure to hyposmotic extracellular solution resulted in its reversible activation. The induced current was due to Cl‐, based on its reversal close to the chloride equilibrium potential and sensitivity to blockade by Cl‐ channel inhibitors. The hyposmotically induced current could be activated in Ca(2+)‐free solutions containing 0.2 mM EGTA. 4. When studied in the current‐clamp configuration, hyposmotic stimulation caused depolarization from ‐76 +/‐ 5 to ‐5 +/‐ 6 mV (mean +/‐ S.D., n = 7). 5. Unitary Cl‐ currents were recorded in the cell‐attached patch configuration at positive potentials. Single channels had a slope conductance of 19 +/‐ 3 pS (n = 5). Reduction of the external [Cl‐] shifted the current‐voltage relationship in the positive direction, supporting the conclusion that these were Cl‐ currents. Like the whole‐cell currents, single channel Cl‐ currents were activated by exposure of cells to hyposmotic bathing solution. 6. We conclude that rabbit osteoclasts express an outwardly rectifying Cl‐ current that can be activated by osmotic stress. Cl‐ channels may play a role in cell volume regulation and may also provide conductive pathways for dissipating the potential difference that arises from electrogenic proton transport during bone resorption.

Section: Comparison With Cl-currents In Other Cellsmentioning

confidence: 99%

Outwardly rectifying chloride current in rabbit osteoclasts is activated by hyposmotic stimulation.

Kelly

Dixon

Sims

1994

“…In excised inside-out patches, application of the catalytic subunit of PKA to the bath solution (internal surface of the membrane) did not activate the channel. The gating kinetics of our described PKC-activated Clchannel show slower bursting behaviour than that in the epithelial Cl-channels (Garcia-Diaz, 1991;Suzuki et al 1991). Mean open lifetimes in the epithelial Cl-channels are within several milliseconds (Garcia-Diaz, 1991;Suzuki et al 1991), indicating the rapid gating during a burst.…”

Section: Discussionmentioning

confidence: 70%

“…The gating kinetics of our described PKC-activated Clchannel show slower bursting behaviour than that in the epithelial Cl-channels (Garcia-Diaz, 1991;Suzuki et al 1991). Mean open lifetimes in the epithelial Cl-channels are within several milliseconds (Garcia-Diaz, 1991;Suzuki et al 1991), indicating the rapid gating during a burst. In contrast, the cyclic AMP-regulated Cl-channel, recently reported in guinea-pig cardiac cells, shows several similarities to our described channel (Ehara & Matsuura, 1993).…”

Section: Discussionmentioning

confidence: 70%

Activation of the plasma membrane chloride channel by protein kinase C in isolated guinea‐pig hepatocytes.

Koumi

Satō

Aramaki

1995

1. To assess the nature of the underlying mechanism of noradrenaline-induced increase of Clconductances in hepatocytes, macroscopic and unitary currents through noradrenalineinduced Cl-channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, cell-attached and excised inside-out configurations of the patch-clamp technique. 2. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]1) was set at 0 1 uM, bath application of noradrenaline activated the time-independent membrane currents under whole-cell voltage-clamp conditions. The current was similarly activated by phorbol ester (PMA), an activator of protein kinase C (PKC), while a specific protein kinase C inhibitor, H-9, reversed PMA activation of the current. The inactive phorbol ester, 4a-phorbol 12-myristate, 13-acetate (aPMA), failed to activate the channel. 3. The reversal potential of the PMA-activated current shifted by -60 mV per 10-fold change in the external Cl-concentration, indicating that the current was Cl-selective. Bath application of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) partially inhibited both the noradrenaline-and PMA-induced currents. 4. In single channel recordings from cell-attached patches, bath application of noradrenaline or PMA induced unitary current activity, the averaged slope conductance of which was 10 1 + 1P5 pS (mean + S.D.; n = 12) in the noradrenaline-induced current and 9-7 + 1-3 pS (n = 7) in the PMA-induced current. The open time distribution was moderately well fitted by a single exponential function with mean open lifetime of 88-5 + 10-6 ms (n = 10), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 24 4 + 5 8 ms (n = 10) and for the slow component of 316 9 + 49-2 ms (n = 10). 5. Bath application of purified PKC to excised inside-out patches activated the channel. The PKC selective inhibitor, , and DIDS inhibited the PKC-activated channel. 6. These results suggest that PKC can phosphorylate the channel protein or a related structure leading to the activation of Cl-channels in guinea-pig hepatocytes.

“…Quinidine has been shown to block K+ channels in a variety of cell types (Merot et al 1989;Garcia-Diaz, 1991 (Kehl, 1991;Synders, Knoth, Roberds & Tamkun, 1992). However, in contrast to that seen in a number of voltage-gated K+ channels (Kehl, 1991;Synders et al 1992) inhibition was not voltage dependent.…”

Section: Characteristics Of Gbamentioning

confidence: 99%

Two K(+)‐selective conductances in single proximal tubule cells isolated from frog kidney are regulated by ATP.

Robson

Hunter

1997

1. The whole-cell and single channel patch clamp techniques were used to identify K+-selective conductances in single proximal tubule cells isolated from frog kidney and to examine their ATP sensitivity. Whole-cell currents were inhibited by the K+ channel inhibitors Ba2P and quinidine in a dose-dependent manner. Addition of Ba2+ alone, quinidine alone, or both inhibitors together revealed two separate conductances, one of which was blocked by both Ba2P and quinidine (GBa), the other being sensitive to quinidine alone (Gquin).2. With Na+-containing Ringer solution in the bath and K+-containing Ringer solution in the pipette, both currents were selective for K+ over Nae. The K+: Na+ selectivity ratio of GBa was around 50: 1, while that of Gquin was 4: 1. In symmetrical KCl solutions GBa showed inward rectification, while Gquin demonstrated outward rectification. 3. In the absence of pipette ATP, both GBa and Gquin ran down over 10 min. However, when 2 mm ATP was included in the pipette GBa increased, while Gquin remained unchanged. 4. Single channel studies demonstrated that a basolateral K+ channel shared several of the characteristics of GBa. It was inhibited by both Ba2P and quinidine, underwent run-down in excised patches in the absence of ATP, and was activated by ATP.