Intracellular calcium changes trigger connexin 32 hemichannel opening

Abstract

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.

Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters

“…If the mechanism of action of the peptides involved a gating process one might expect the contrary, opening of the connexin/pannexin channels, assuming the peptides with extracellular loop sequences were to mimic the action of the docking process in gap junction channel formation. The rationale for the use of 32 Gap24, which has the sequence of cytoplasmic aspects of connexin32, is even more mysterious (14). The high peptide concentrations required for significant effects on channel currents as shown here or on downstream events such as ATP release or calcium wave Fig.…”

“…The use of the peptides 32 Gap24, 43 Gap26, 43 Gap 27, and 10 Panx1 have been published (14,18,36). Their sequences are GHGDPLHLEEVKC ( 32 Gap24), VCYDKSFPISHVR ( 43 Gap26), SRPTEKTIFII ( 43 Gap 27), and WRQAAFVDSY ( 10 Panx1).…”

“…From the attenuation of the measured parameters, it was inferred that the peptides inhibited rather than activated gap junction hemichannels, that is, connexons. While most of the peptides used in these studies contained extracellular loop sequences, recently, it was reported that Gap 24, a peptide with sequence contained in the cytoplasmic loop, was more effective in attenuating the measured parameters (14). The theoretical framework for such an action is not clear.…”

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries

“…While moderate ( 500 nM) [Ca 2þ ] i elevation stimulates hemichannel opening, most likely via calmodulin signalling, [6][7][8] larger (500-1000 nM) [Ca 2þ ] i increases result in hemichannel closure, presumably via actomyosin contraction that disrupts loop-tail interaction. 37 Our unitary current data demonstrate that CT9 prevents high [Ca 2þ ] i closure of Cx43-hemichannels.…”

“…Hemichannels are permeable to Ca 2þ and may thus contribute to cellular Ca 2þ entry 4 and diffusive ATP release. 5 Moreover, hemichannel opening is [Ca 2þ ] i -dependent in a bimodal manner, [6][7][8][9][10] with moderate [Ca 2þ ] i elevation favouring opening and above $500 nM [Ca 2þ ] i inhibiting opening. The bimodal Ca 2þ -dependence of hemichannels resembles the Ca 2þ -dependence of inositol-trisphosphate receptor (IP 3 R) channels, which are Ca 2þ release channels in the endoplasmic reticulum (ER) that play a central role in generating Ca 2þ oscillations in diverse cell types including SMCs.…”