Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

Abstract

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.

Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3)

“…The release of ATP from HLE-B3 cells was evaluated as previously described (Figueroa et al, 2014). Briefly, cells were seeded into 60-mm diameter dishes at 70% confluence and 48 h later they were washed once with DCFS and then 500 µl of the same fresh solution were added.…”

“…Previously, we have shown that LA induces opening of hemichannels formed by both Cx46 in Xenopus laevis oocytes, and by Cx43 in HeLa cells (Retamal et al, 2011;Figueroa et al, 2014). To test whether LA increases hemichannels activity in HLE-B3 cells, we determined the effects of acute exposure to this fatty acid using the Etd + uptake assay.…”

Connexins and Pannexins in Bone and Skeletal Muscle

“…43 and 40.1 (ortholog of mouse Cx39) [85], suggesting that these proteins could play a relevant role in these pathological conditions. Interestingly, streptomycin has been found to reduce stretch-induced membrane permeability in mdx muscles [97] and is also known to block connexin hemichannels [98]. …”

A Novel E. Coli-Based Assay for Rapid Screening of Hemichannel Function

“…Inhibitors of connexin-formed channels with affinities in the low-nM range are not available, but these compounds at the concentrations employed are known to inhibit HCs [80-83]. Although non-specific, divalent cations are well-known inhibitors of HCs [84]; 2) The sensitivity of growth complementation by Cx26 mutants to the inhibitory effect of Ca 2+ was altered [79].…”