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1From the Laboratory of Neurophysiology, Department of Physiological Sciences I, Institute of Biomedical Investigations August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Barcelona, Spain; and the 3Internal Medicina Service, IDIBAPS, Hospital Clinic, Barcelona, Spain.
PURPOSE. Anterior chamber structures are subjected to changes in intraocular pressure (IOP). Several studies have pointed out that trabecular meshwork (TM) cells are sensitive to mechanical stretch and that cell-signaling mechanisms are activated in response to elevated pressure. Because membrane stretch has been shown to be a modulator of several ionic conductances, this study was conducted to determine its effects on the high-conductance Ca2+-activated K+ (BKCa) channels present in TM cells.
Methods
Primary cultures of TM cells from bovine eyes were used. Patch-clamp recordings were performed in the cell-attached, inside-out, and whole-cell configurations. To stretch the cell membrane, both suction to the rear end of the patch pipette and hypotonic shock were used. Intracellular calcium concentration ([Ca2+]i) was measured in TM cells loaded with fura-2, using an epifluorescence microscope coupled to a charge-coupled device (CCD) camera.
Results
Electrophysiological characterization of BKCa channels was in agreement with previous studies. In cell-attached patches, the open probability of the BKCa channel (i.e., the amount of time the channel is open) increased consistently when 14- to 45-mm Hg suctions were applied at a constant depolarized voltage. At a constant pressure (25 or 45 mm Hg), channel openings increased when depolarizing pulses were applied to the patch. Stretch activation of the BKCa channel was not mediated by increases in [Ca2+]i, because it was present in inside-out patches maintained at a constant Ca2+ concentration. Nevertheless, it cannot be ruled out that at low suction levels, a minimum Ca2+ concentration is necessary for channel activation. Whole-cell currents carried by BKCa channels increased when the isotonic solution in the bath was exchanged with a hypotonic solution and were selectively blocked by iberiotoxin. In our conditions, the hypotonic shock did not modify [Ca2+]i.
Conclusions
The data show that in TM cells, open probability of the BKCa channel is enhanced by membrane stretching as well as by membrane depolarization and [Ca2+]i. Changes in membrane tension induced by cell volume increase also activated whole-cell BKCa currents. Homeostatic mechanisms in TM cells may involve BKCa channel activation in response either to changes in cell volume or changes in IOP.
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