Cyclic nucleotide permeability through unopposed connexin hemichannels

Abstract

Cyclic adenosine monophosphate (cAMP) is a well-known intracellular and intercellular second messenger. The membrane permeability of such molecules has potential importance for autocrine-like or paracrine-like delivery. Here experiments have been designed to demonstrate whether gap junction hemichannels, composed of connexins, are a possible entrance pathway for cyclic nucleotides into the interior of cells. HeLa cells stably expressing connexin43 (Cx43) and connexin26 (Cx26) were used to study the cyclic nucleotide permeability of gap junction hemichannels. For the detection of cAMP uptake, the cells were transfected using the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH) as the cAMP sensor. SpIH derived currents (Im) were recorded in whole-cell/perforated patch clamp configuration. Perfusion of the cells in an external K+ aspartate- (KAsp) solution containing 500 μM cAMP and no extracellular Ca2+, yielded a five to sevenfold increase in the Im current level. The SpIH current increase was associated with detectable hemichannel current activity. Depolarization of cells in Ca2+-free NaCl perfusate with 500 μM cAMP also induced a SpIH current increase. Elevating extracellular Ca2+ to mM levels inhibited hemichannel activity. Perfusion with a depolarizing KAsp solution containing 500 μM cAMP and 2 mM Ca2+ did not increase SpIH currents. The addition of the gap junction blocker carbenoxolone to the external solution inhibited cAMP uptake. Both cell depolarization and lowered extracellular Ca2+ increase the open probability of non-junctional hemichannels. Accordingly, the SpIH current augmentation was induced by the uptake of extracellular cAMP via open membrane hemichannels in Cx43 and Cx26 expressing cells. The data presented here show that hemichannels of Cx43 and Cx26 are permeable to cAMP, and further the data suggest that hemichannels are, in fact, a potential pathway for cAMP mediated cell-to-cell signaling.

Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

“…It is, at present, not clear why different current patterns are obtained in astrocytes exposed to DCFS, Cx43-expressing HeLa cells, and Xenopus oocytes, but differences in electrophysiological approaches may play a role (sharp electrode versus fast whole cell patch versus perforated whole cell patch). The astrocytic data of this study are in agreement with data from Cx43-expressing HeLa cells and Xenopus oocytes, thus showing a lack of significant Cx43-mediated DCFS-induced current at negative membrane potentials (4,28,30). In rat astrocytes, we detected a DCFS-induced increase in uptake of ethidium and Yo-Pro which was, however, insensitive to Gd 3ϩ and therefore unlikely to be Cx43-mediated.…”

“…Although several of these large pore channels serve as dye/ ATP conduits as well as ion channels, some channels exhibit a noticeable disconnect between the ability to pass dyes/ATP and current; although dye uptake and ATP/glutamate/cAMP permeability can be detected at physiologically relevant membrane potentials in the negative range (4,9,(27)(28)(29), membrane conductance for Px1 and Cx43 appears predominantly at positive membrane potentials (1,4,9,25). Prolonged exposure to positive membrane potentials may even be required to detect Cx43-mediated membrane conductance (26).…”

Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics

“…These data suggest that Cx43 hemichannels are impermeable to atomic ions at physiologically relevant membrane potentials in the negative range (or the low positive range up to +30 mV experienced by the cardiomyocyte Cx43 during the cardiac action potential); ion permeation events, albeit with very low open probability, only occur at highly positive potentials [103]. Similar results in Cx43-expressing HeLa cells or cardiomyocytes were later obtained by other research groups [64,104] confirming the negligible Cx43-mediated current activity at physiologically relevant membrane potentials. A single study in cultured astrocytes reported current activity upon removal of divalent cations in three out of the five tested cells, with the two remaining cells displaying no divalent cation-sensitive current activity [49].…”

“…Notably, ATP with its negative charges produces a current when traversing a membrane [42]. It therefore remains puzzling with the multitude of studies reporting Cx43-mediated ATP release at physiological membrane potentials [42,53,67,98,115], at which no Cx43-mediated conductance occurs [52, 64,103,104]. Several other molecules have been suggested to permeate connexins in their hemichannel configuration, i.e., glutathione, glutamate, and prostaglandins [12, 41, 74, 119-121], although glutamate release has, in addition, been assigned to other channel types in astrocytes [122,123] and microglia [124].…”

Regulation of Vascular and Renal Function by Metabolite Receptors

“…During the receptor-mediated heightened activity of AC, some intracellular cAMP is released into the extracellular milieu through multidrug resistance gene products or connexin hemichannels (103–105). The released cAMP is first converted to AMP by ectophosphodiesterases and/or tissue-nonspecific alkaline phosphatases.…”