Biophysical and pharmacological characterization of hypotonically activated chloride currents in cortical astrocytes

Abstract

Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge. This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels. Using whole-cell recordings, we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge. These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions: I ->NO 3 ->Cl ->Gluc -≥MeS ->Ise -. Interestingly, extracellular I -, albeit showing the greatest permeability, blocked the currents with an IC 50 of ≈50 mM. Currents were almost completely inhibited by 123 μM NPPB and partially inhibited by 200 μM niflumic acid or 200 μM DIDS. Additionally, the total number of Cl -ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation. We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes. To examine potential candidate chloride channel genes expressed by astrocytes, we employed RTPCR to demonstrate the presence of transcripts for ClC-2, 3, 4, 5, and 7, as well as for VDAC and CFTR in cultured astrocytes. Moreover, we performed immunostaining with antibodies against each of these channels and showed the strongest expression of ClC-2 and ClC-3, strong expression of ClC-5 and VDAC, weak expression of ClC-7 and very weak expression of ClC-4 and CFTR. Intriguingly, although we found at least seven Cl -channel proteins from three different gene families in astrocytes, none appeared to be active in resting cells.

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes

“…In addition, ClC-3, ClC-4, and ClC-7 currents are sensitive to DIDS whereas ClC-2 and ClC-5 currents are resistant to DIDS (Li and Weinman, 2002;Parkerson and Sontheimer, 2004). In rat articular cartilages, there is mRNA expression of ClC-3 (Ponce et al, 2012).…”

“…The ClC gene family contains nine members coding voltage-dependent Cl 2 channels (Jentsch et al, 2002;Li and Weinman, 2002;Parkerson and Sontheimer, 2004). ClC-2 currents exhibit an inward rectification, whereas ClC-4, ClC-5, and ClC-7 currents show strong outward rectification.…”

“…Statistical significance is indicated as *P , 0.05 or **P , 0.01 versus normal medium (350 mOsm). Parkerson and Sontheimer, 2004). ClC-2 and ClC-7 channels are activated by acidic pH whereas ClC-5 currents are inhibited by acidic pH.…”

“…ClC-2 and ClC-7 channels are activated by acidic pH whereas ClC-5 currents are inhibited by acidic pH. However, the pH sensitivities of ClC-3 and ClC-4 vary with the type of cells (Kawasaki et al, 1999;Diewald et al, 2002;Parkerson and Sontheimer, 2004;Matsuda et al, 2008Matsuda et al, , 2010.…”

Optically Recorded Response of the Superficial Dorsal Horn: Dissociation From Neuronal Activity, Sensitivity to Formalin-Evoked Skin Nociceptor Activation

“…Glial activation involves changes in astrocyte shape and volume and altered communication between neighboring astrocytes via gap junctions (Meme et al 2004;Olsen and Sontheimer 2004;Walz 2002). Astrocytes in the affected region of the dorsal horn exhibit increased expression of glial fibrillary acidic protein (GFAP) (Garrison et al 1991), decreased expression of glutamate transporters and K IR -type membrane potassium channels (Huang et al 2004;Kawahara et al 2002;MacFarlane and Sontheimer 1997;Olsen and Sontheimer 2004), increased Cl Ϫ conductance (Parkerson and Sontheimer 2004;Walz 2002), absence of the [Ca 2ϩ ] i transients normally evoked by afferent activity (Aguado et al 2002), and altered expression of genes regulating production and release of neuroactive cytokines, chemokines, growth factors, and NO (Meeuwsen et al 2003;Milligan et al 2001).…”