Indirectly Gated Cl−-Dependent Cl−Channels Sense Physiological Changes of Extracellular Chloride in the Leech

The maintenance of ion homeostasis requires adequate ion sensors. In leeches, 34 nephridial nerve cells (NNCs) monitor the Cl− concentration of the blood. After a blood meal, the Cl−concentration of leech blood triples and is gradually restored to its normal value within 48 h after feeding. As previously shown in voltage-clamp experiments, the Cl− sensitivity of the NNCs relies on a persistent depolarizing Cl− current that is turned off by an increase of the extracellular Cl− concentration. The activation of this Cl−-dependent Cl− current is independent of voltage and of extra- and intracellular Ca2+. The transduction mechanism is now characterized on the single-channel level. The NNC's sensitivity to Cl− is mediated by a slowly gating Cl−-dependent Cl−channel with a mean conductance of 50 pS in the cell-attached configuration. Gating of the Cl− channel is independent of voltage, and channel activity is independent of extra- and intracellular Ca2+. Channel activity and the macroscopic current are reversibly blocked by bumetanide. In outside-out patches, changes of the extracellular Cl− concentration do not affect channel activity, indicating that channel gating is not via direct interaction of extracellular Cl− with the channel. As shown by recordings in the cell-attached configuration, the activity of the channels under the patch is instead governed by the Cl− concentration sensed by the rest of the cell. We postulate a membrane-bound Cl−-sensing receptor, which—on the increase of the extracellular Cl− concentration—closes the Cl− channel via a yet unidentified signaling pathway.

Blockade of Adenosine Triphosphate–sensitive Potassium Channels by Thiamylal in Rat Ventricular Myocytes

Background The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. Methods Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1). Results In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. Conclusions The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.

Chloride channels in the eel intestine

Patches of freshly isolated epithelial cells from eel Anguilla anguilla intestine bathed by the same solution on both sides in the cell-attached configuration had conductances of 57.0+/−1.8 pS (for positive voltages) and 13.3+/−0.7 pS (for negative voltages) (means +/− s.e.m., N=25). Electrical activity was spontaneous in the cell-attached configuration, but was frequently lost after excision. In inside-out patches, channel activity was restored by strong hyperpolarization (−150 mV for 5 s) or depolarization (+150 mV for 5 s). Channel activity was inhibited by the Cl- transport blocker DIDS (1 mmol l-1). The membrane potential measured using the nystatin slow whole-cell technique in primary cultured eel intestine epithelial cells was −35.4+/−1.0 mV (N=14), similar to the expected equilibrium potential for Cl- (−38.2 mV). Removal of Cl- from the bath or application of DIDS caused 16 mV and 6–7 mV depolarizing shifts in reversal potential, respectively. In one experiment, DIDS also induced a reduction in cell conductance from 0. 011+/−0.014 to 0.002+/−0.005 nS. The addition of 0.5 mmol l-1 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (a membrane-permeable analogue of cyclic AMP) to the bath caused an increase in conductance without affecting the reversal potential.