Abstract
Epithelial cells express calcium-activated Cl(-) channels of unknown molecular identity. These Cl(-) channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. The family of bestrophins has been suggested to form calcium-activated Cl(-) channels. Here, we demonstrate molecular and functional expression of bestrophin-1 (BEST1) in mouse and human airways, colon, and kidney. Endogenous calcium-activated whole cell Cl(-) currents coincide with endogenous expression of the Vmd2 gene product BEST1 in murine and human epithelial cells, whereas calcium-activated Cl(-) currents are absent in epithelial tissues lacking BEST1 expression. Blocking expression of BEST1 with short interfering RNA or applying an anti-BEST1 antibody to a patch pipette suppressed ATP-induced whole cell Cl(-) currents. Calcium-dependent Cl(-) currents were activated by ATP in HEK293 cells expressing BEST1. Thus, BEST1 may form the Ca2+-activated Cl(-) current, or it may be a component of a Cl(-) channel complex in epithelial tissues.
Highlights
Molecular candidates for CaCC have been proposed in the past
Expression of BEST1 was reported to be limited to the retinal pigment epithelium, it has been detected in cultured airway epithelial cells [15, 16]
CaCC have been found in primary murine nasal cell cultures and in cystic fibrosis nasal epithelium but not in normal murine nasal tissue (20 – 22)
Summary
Molecular candidates for CaCC have been proposed in the past. A family of Ca2ϩ-activated ClϪ channels (CLCA) has been identified [8, 9]. Expression of BEST1 was reported to be limited to the retinal pigment epithelium, it has been detected in cultured airway epithelial cells [15, 16]. We will provide evidence that endogenously expressed BEST1 causes Ca2ϩ-activated ClϪ conductance in epithelial tissues. Detection of Mouse and Human BEST1 Proteins by Western Blotting—Protein was isolated from mouse nose, trachea, and kidney epithelium and from cultured cells using buffer containing 150 mmol of NaCl, 50 mmol of Tris, 100 mmol of dithiothreitol, 1% Nonidet P-40, and 1% protease inhibitor mixture (Sigma).
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