Gap junction hemichannels in astrocytes of the CNS

Abstract

Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to approximately 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD+ and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca2+ and intracellular H+ ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca2+, and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.

Figure 1

Conditions that open or close hemichannels. Connexins are tetraspan proteins with both amino and carboxy terminals on the cytoplasmic side of the membrane (inset, upper right). Each hemichannel contains six connexin molecules and under physiological conditions are preferentially closed (left). Open probability of hemichannels is increased in low [Ca²⁺]_O, at positive membrane potentials (+V_m), by such pharmacological agents as quinine and quinidine, and after metabolic inhibition leading to dephosphorylation and increase in reactive oxygen species (ROS) and generation of as yet unidentified cytoplasmic signals. Open probability is decreased by high [Ca²⁺]_O, negative membrane potentials (−V_m), low intracellular pH, phosphorylation of cytoplasmic domains, polyvalent cations, including Co²⁺, Ni²⁺, Mg²⁺, La²⁺ and Gd³⁺, and low intracellular pH and other gap junction blockers.

Figure 2

Time course of ethidium bromide (EtdBr) and propidium iodide (PI) uptake in metabolically inhibited astrocytes. Confluent cultures of rat cortical astrocytes were treated with iodoacetic acid and antimycin A and the time course of EtdBr and IP uptake was measured (in separate experiments). EtdBr uptake began a few minutes after application of metabolic inhibitors (at 60 min). PI uptake was minimal until ~ 130 min of metabolic inhibition at which time uptake increased abruptly. Other experiments indicate that this delayed uptake is because of the loss of membrane integrity.

Figure 3

Ethidium bromide uptake by astrocytes treated with metabolic inhibitors is blocked by 18-glycyrrhetinic acid. Four hours after plating at a very low density (200 cells/60 mm diameter culture dish) cortical rat astrocytes were exposed to iodoacetic acid plus antimycin A for 75 min or kept in control medium. Ethidium bromide (50 µm) was then applied for 2 min to cells in the control medium (a), to metabolically inhibited cells (b), and to metabolically inhibited cells treated with 75 µm 18β-glycyrrhetinic acid for 5 min before the dye application (c). Cells treated only with metabolic inhibitors were stained (b), and cells under control conditions or treated with metabolic inhibitors plus the gap junction blocker remained unstained; (d), (e) and (f) are phase contrast views of the fluorescent views showed in (a), (b) and (c), respectively. Bar 20 µm.