Characterization of potassium and chloride channels in the basolateral membrane of bovine nonpigmented ciliary epithelial cells.
OBJECTIVE
The pigmented epithelial (PE) and the nonpigmented epithelial (NPE) cells of the ciliary body may function as a syncytium for secretion of the aqueous humor, with solute and water entering through the PE and leaving through the NPE cell. Secretion across the basolateral membrane of the NPE cell was postulated to depend on coupling sodium extrusion through the NaK-ATPase to efflux through Cl channels. In the current study, the authors used single-channel patch clamp techniques to characterize the ion channels present in the basolateral membrane of the NPE cell.


METHODS
Mild enzymatic digestion of the bovine ciliary process was used to obtain pairs or clusters of NPE cells that were coupled to their neighbor PE cells. Cells were dispersed immediately onto a coverslip, bathed in a HEPES-buffered saline, and imaged with an inverted microscope. With the anatomic relationship between cells still intact, a patch-pipette electrode was applied to the basolateral surface of the NPE cell. Single-channel currents were then characterized in 52 cells by using either the cell-attached or excised versions of the patch-clamp technique.


RESULTS
A calcium-dependent "maxi" K channel was found in approximately 33% of the patches. It was activated by depolarizing voltage steps in cell-attached patches and was relatively inactive near the resting potential. When excised from the cell, it was activated by high levels of bath calcium and, in symmetrical K solutions (150 mM), showed a linear current-voltage (I-V) relationship with a slope conductance of approximately 150 pS. This channel was highly selective to K over Na and was blocked by barium (2 mM) or quinine (1 mM). In cell-attached recordings, a smaller conductance Cl channel was observed in 73% of the patches. The channel currents were inward at resting potential and outward with depolarizing voltage steps, with a pipette reversal potential of approximately -23 mV. The current-voltage relationship of this channel was nearly linear with a slope conductance of approximately 24 pS. When pipette Na and K were replaced with N-methyl-D-glucamine, this channel produced an inward current showing a high selectivity for Cl. This Cl-selective channel was activated by dibutyryl-cyclic adenosine monophosphate (cAMP) but not by elevation of intracellular calcium with ionomycin or by anisosmotic cell swelling.


CONCLUSIONS
Two ion channels were found in the NPE basolateral membrane: One was K selective, the other was Cl selective. The cAMP-activated Cl channel may be important in supplying the counterion for Na extrusion across the basolateral surface of the NPE cell, whereas the calcium-dependent maxi K channel may be useful for membrane hyperpolarization to increase the driving force for Cl exit.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000032:analysis* / Q000187:drug effects; D002924:Ciliary Body / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D004553:Electric Conductivity; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000032:analysis* / Q000187:drug effects
D018118:Chloride Channels; D015221:Potassium Channels; D000242:Cyclic AMP; D002118:Calcium