Mariana C. Fiori

BACKGROUND: Indirect evidence suggests that connexin hemichannels are permeable to Ca(2+), but direct demonstration is lacking. RESULTS: Calcium moves into liposomes containing purified Cx26 in response to a concentration gradient. CONCLUSION: Cx26 hemichannels are permeable to Ca(2+). SIGNIFICANCE: Cx26 hemichannels may play a role in Ca(2+) influx into cells under conditions that lead to hemichannel activation, such as ischemic damage. Gap junction channels communicate the cytoplasms of two cells and are formed by head to head association of two hemichannels, one from each of the cells. Gap junction channels and hemichannels are permeable to ions and hydrophilic molecules of up to M(r) 1,000, including second messengers and metabolites. Intercellular Ca(2+) signaling can occur by movement of a number of second messengers, including Ca(2+), through gap junction channels, or by a paracrine pathway that involves activation of purinergic receptors in neighboring cells following ATP release through hemichannels. Understanding Ca(2+) permeation through Cx26 hemichannels is important to assess the role of gap junction channels and hemichannels in health and disease. In this context, it is possible that increased Ca(2+) influx through hemichannels under ischemic conditions contributes to cell damage. Previous studies suggest Ca(2+) permeation through hemichannels, based on indirect arguments. Here, we demonstrate for the first time hemichannel permeability to Ca(2+) by measuring Ca(2+) transport through purified Cx26 hemichannels reconstituted in liposomes. We trapped the low affinity Ca(2+)-sensitive fluorescent probe Fluo-5N into the liposomes and followed the increases in intraliposomal [Ca(2+)] in response to an imposed [Ca(2+)] gradient. We show that Ca(2+) does move through Cx26 hemichannels and that the permeability of the hemichannels to Ca(2+) is high, similar to that for Na(+). We suggest that hemichannels can be a significant pathway for Ca(2+) influx into cells under conditions such as ischemia.

Styrene-maleic acid copolymers allow for solubilization and reconstitution of membrane proteins into nanodiscs. These polymer-encased nanodiscs are promising platforms for studies of membrane proteins in a near-physiologic environment without the use of detergents. However, current styrene-maleic acid copolymers display severe limitations in terms of buffer compatibility and ensued flexibility for various applications. Here, we present a new family of styrene-maleic acid copolymers that do not aggregate at low pH or in the presence of polyvalent cations, and can be used to solubilize membrane proteins and produce nanodiscs of controlled sizes.

Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.

Gap-junction channels (GJCs) are aqueous channels that communicate adjacent cells. They are formed by head-to-head association of two hemichannels (HCs), one from each of the adjacent cells. Functional HCs are connexin hexamers composed of one or more connexin isoforms. Deafness is the most frequent sensineural disorder, and mutations of Cx26 are the most common cause of genetic deafness. Cx43 is the most ubiquitous connexin, expressed in many organs, tissues, and cell types, including heart, brain, and kidney. Alterations in its expression and function play important roles in the pathophysiology of very frequent medical problems such as those related to cardiac and brain ischemia. There is extensive information on the relationship between phosphorylation and Cx43 targeting, location, and function from experiments in cells and organs in normal and pathological conditions. However, the molecular mechanisms of Cx43 regulation by phosphorylation are hard to tackle in complex systems. Here, we present the use of purified HCs as a model for functional and structural studies. Cx26 and Cx43 are the only isoforms that have been purified, reconstituted, and subjected to functional and structural analysis. Purified Cx26 and Cx43 HCs have properties compatible with those demonstrated in cells, and present methodologies for the functional analysis of purified HCs reconstituted in liposomes. We show that phosphorylation of serine 368 by PKC produces a partial closure of the Cx43 HCs, changing solute selectivity. We also present evidence that the effect of phosphorylation is highly cooperative, requiring modification of several connexin subunits, and that phosphorylation of serine 368 elicits conformational changes in the purified HCs. The use of purified HCs is starting to provide critical data to understand the regulation of HCs at the molecular level.

Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or "free HCs" at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.