The role of ClC‐3 in volume‐activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition

Abstract

A chloride current with mild outward rectification was induced in the native bovine non‐pigmented ciliary epithelial (NPCE) cells by a 23 % hypotonic solution. The current showed no or little inactivation at depolarized steps. ATP blocked 88 and 61 % of the outward and inward components of the volume‐activated chloride current (ICl,vol) with an IC50 of 5.3 and 9.6 mM, respectively. The volume‐activated chloride current was decreased and the activation of the current was delayed by inhibiting endogenous ClC‐3 expression using a ClC‐3 antisense oligonucleotide. The inhibition of the current as a function of antisense concentration was asymptotic with a maximum about 60 %. The remaining current was probably not derived from ClC‐3 and was inhibited by ATP. ClC‐3 expression in the bovine NPCE cells was verified by immunofluorescence studies. ClC‐3 immunofluorescence was distributed throughout the cells but with the predominant location within the nucleus. The expression of ClC‐3 protein was diminished by the ClC‐3 antisense oligonucleotide with the greatest diminution occurring in the nuclear region. The size of the volume‐activated chloride current was positively correlated with the ClC‐3 immunofluorescence level. Regulatory volume decrease of the NPCE cells was reduced by ClC‐3 antisense oligonucleotide. We conclude that endogenous ClC‐3 is associated with the volume‐activated chloride current and is involved in cell volume regulation, but that it can only contribute towards a proportion of the current in NPCE cells. The nuclear predominance of ClC‐3 immunofluorescence in NPCE cells, the absence of basal activity of chloride current and the marked pharmacological differences between IClC‐3 and ICl,vol argue against ClC‐3 being the only, or even the main, volume‐activated chloride channel in NPCE cells.

Chloride channel ClC-3 in gills of the euryhaline teleost,Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption

“…Generally, hyposmotic shock can result in cell swelling (Stutzin and Hoffmann, 2006). Meanwhile, ClC-3, a member of the CLC family, has been reported to be activated by cell swelling (Duan et al, 1997;Duan et al, 1999), and several studies have suggested that ClC-3 represents a major molecular entity responsible for native volume sensing in outwardly rectifying anion channels of various cell types in hyposmotic media (Wang et al, 2000;Duan et al, 2001;Hermoso et al, 2002).…”

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel

“…The whole-cell membrane current of a single cell was amplified through an EPC-9 amplifier (HEKA Elektronik Dr. Schulze GmbH, Lambrecht, Germany), and via filtering by 2.9 kHz, the currents were recorded to the Pulse and Pulsefit software system (HEKA Elektronik Dr. Schulze GmbH). Whole-cell patch clamp recording parameters were as follows (11,17): Under the voltage clamp mode, the cells were clamped at 0, ±40 and ±80 mV. Each clamp pulse was held for 200 msec with an interval gap of 4 sec.…”

“…In fact, studies have shown that numerous ion channels [such as Ca 2+ channels (8) and K + channels (9)] have a role in osteogenic differentiation. ClC-3 is a subtype of the volume-sensitive chloride channel family that has a role in cell volume regulation (10)(11)(12), as well as in cell proliferation (13), apoptosis (10), cell cycle progression (14) and intracellular acidification regulation in the osteoclast (15). Studies have also reported the involvement of chloride channels in the metabolism of osteoblasts, for example, ClC-3 protein expression can promote osteogenesis via Runx2 gene expression in mouse osteoblasts (16).…”

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

“…ClC-3 was expressed in CNE-2Z cells and has been proposed by others and us to be the molecular candidate involved in the activation or regulation of volume-sensitive Cl Ϫ currents (7,9,18,28,31). To investigate the role of ClC-3 in the activation of the acid-activated chloride currents, the effect of knockdown of ClC-3 expression by ClC-3 siRNA was tested.…”

“…Analysis of current properties indicates that there are some similarities between the acid-induced Cl Ϫ current and the volume-sensitive Cl Ϫ current. ClC-3 has been proposed by others and us to be the molecular candidate involved in the activation or regulation of volume-sensitive Cl Ϫ current (7,9,18,28). The role of ClC-3 proteins in activation of acidinduced Cl Ϫ current is not clear.…”