Stable and functional expression of the CIC-3 chloride channel in somatic cell lines

Abstract

The CIC family is the superfamily of voltage-gated Cl- channels. Although the CIC channels expressed in Xenopus oocytes have been characterized, their channel properties are still poorly understood. We recently cloned a unique member of the CIC family, CIC-3, that is expressed abundantly in neurons. Its channel activity was regulated by phorbol esters. Now, we have established a stably transfected somatic cell line expressing functional CIC-3 channels and examined the CIC-3 single-channel current by patch-clamp techniques. In inside-out patches from the stably transfected cells, a rise of bath Ca2+ concentration in the physiological range of intracellular Ca2+ concentrations inhibited the CIC-3 single-channel currents. This inhibition by Ca2+ was independent of phosphorylation and ATP. Thus, the CIC-3 channel is a Ca(2+)-sensitive Cl- channel localized in neuronal cells, and its Ca2+ sensitivity implies a physiological role in neuronal functions.

Biophysical Properties of ClC-3 Differentiate It from Swelling-activated Chloride Channels in Chinese Hamster Ovary-K1 Cells

“…These currents displayed slight outward rectification, inactivated at positive voltages, and were sensitive to DIDS and phorbol 12,13-dibutyrate. Similar ClC-3 currents were reported by Kawasaki et al (12,20) who described rat ClC-3 currents in oocytes (20) and stably transfected CHO-K1 cells (12). Their currents exhibited somewhat different properties in the different expression systems, but they also showed only a weak outward rectification and sensitivity to DIDS as well as phorbol esters that were similar to that reported by Duan et al (11).…”

“…Heterologous expression of ClC-3 has been reported by two groups (11,12). These groups (11,12) observed large currents in stably transfected cell lines.…”

“…Heterologous expression of ClC-3 has been reported by two groups (11,12). These groups (11,12) observed large currents in stably transfected cell lines. However, the properties of the currents reported are markedly different from those of ClC-4 and ClC-5, the two most closely homologous family members (13).…”

Chloride Channels Regulate HIT Cell Volume but Cannot Fully Account for Swelling-Induced Insulin Secretion

“…This led to the discovery of the CLC superfamily, which includes CLC-0, CLC-1, CLC-2, CLC-3, CLCK1, CLCK2, and other channels (25,66 -70). Although there was early evidence to support the hypothesis that protein-mediating nucleotide-sensitive chloride current (P Cln ) was the ubiquitous outwardly rectifying volume-regulated Cl Ϫ channel (71), it is now believed that CLC-3, which is present in brain, kidney, and heart, among other tissues (25), is the likely molecular counterpart to volume-stimulated Cl Ϫ channels (72). RT-PCR of HIT cell mRNA, using oligonucleotide primers based on the published CLC-3 sequence, isolated a 593-bp transcript that was Ͼ90% homologous to guinea pig, rat, mouse, or human CLC-3.…”

ClC-3 Is a Fundamental Molecular Component of Volume-sensitive Outwardly Rectifying Cl− Channels and Volume Regulation in HeLa Cells and Xenopus laevis Oocytes

“…ClC-2, when expressed, has been shown to display biophysical and pharmacological characteristics that differ from native VSOACs and has also been shown not to contribute to RVD (15,22). ClC-3 was originally cloned from rat kidney by Kawasaki et al (23,24), and, when expressed, currents showed basal activation and inhibition by phorbol esters and Ca 2ϩ (25,26). ClC-3 cloned from guinea pig ventricular myocytes was proposed to be the molecular candidate responsible for cardiac VSOACs (16).…”