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Front Pharmacol
2013 Jun 06;4:75. doi: 10.3389/fphar.2013.00075.
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Cyclic nucleotide permeability through unopposed connexin hemichannels.
Valiunas V
.
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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 (I m) 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 Ca(2) (+), yielded a five to sevenfold increase in the I m current level. The SpIH current increase was associated with detectable hemichannel current activity. Depolarization of cells in Ca(2) (+)-free NaCl perfusate with 500 μM cAMP also induced a SpIH current increase. Elevating extracellular Ca(2) (+) to mM levels inhibited hemichannel activity. Perfusion with a depolarizing KAsp solution containing 500 μM cAMP and 2 mM Ca(2) (+) 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 Ca(2) (+) 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.
FIGURE 1. Immunofluorescent identification of connexin expression in transfected HeLa cells. HeLa cells expressing Cx43 (A) and Cx26 (B) stained with antibodies to Cx43 and Cx26, respectively, show typical punctate staining of Cx43 and Cx26 gap junction plaques at cell–cell contact areas, as well as Cx43 and Cx26 localization in the cell membranes of single cells. Green labeling represents staining of connexin proteins, while blue shows DAPI staining of cell nuclei. The right panels show bright field images. Scale bar, 10 μm.
FIGURE 2. Properties of SpIH channels.
(A) Voltage protocol (Vm) and whole-cell currents (Im) recorded in SpIH transfected HeLaCx43 cells in the absence (B) and presence (C) of 50 μM cAMP in the pipette solution. Schematics in the right panels illustrate whole-cell recording conditions from HeLaCx43/SpIH cells. The NaCl external bath solution contained 2 mM Ca2+ and 500 μM cAMP. (D) Average of tail current densities measured after voltage step to Vm = -100 mV in the absence (6.2 ± 0.9 pA/pF, n = 10 cells) and the presence of intracellular cAMP (31.4 ± 4.3 pA/pF, n = 8 cells), P < 0.001.
FIGURE 3. cAMP induced activation of SpIH channels.
Im elicited by hyperpolarizing pulses (A) (from -20 to -120 mV) in SpIH transfected HeLaCx43 cells in with both 2 mM Ca2+
(B) and with no added Ca2+
(C). Schematics in the right panels of (B) and (C) illustrate the experimental conditions. In both cases the external KAsp bath solution contained 500 μM cAMP. Im increased significantly with Vm and hyperpolarization induced voltage- and time-dependent inward currents when no external Ca2+ was present. (D) Average of current amplitudes recorded in the presence and in the absence of external Ca2+, respectively: 6.4 ± 1.1 pA/pF, n = 10 versus 39.5 ± 5.8 pA/pF, n = 13; P < 0.001.
FIGURE 4. Detection of intracellular cAMP.
(A) Voltage protocol and Im recordings from SpIH transfected HeLaCx43 cells perfused with 2 mM extracellular Ca2+
(B), as well as after the perfusion solution with no Ca2+ added (C) (see schematics on the right for the recording conditions of the cells). (D) Current recorded from the HeLaCx43/SpIH cell in response to voltage pulses from a holding potential of 0 to -100 mV, returning to a tail potential of +50 mV. Over time, perfusion with the Ca2+-free KAsp external solution induced a SpIH current increase to a steady-state. (E) Average of current densities measured in the presence of external Ca2+ and after perfusion with the KAsp solution with no Ca2+: 5.8 ± 0.9 pA/pF versus 37.1 ± 3.6, n = 7, P < 0.001, t-test. 500 μM cAMP was present in the external solution at any time during the experiment.
FIGURE 5. Membrane currents without SpIH expression.
(A)
Im responses from HeLaCx43 cell lacking expression of SpIH to hyperpolarizing pulses recorded in Ca2+-free KAsp solution. (B) Perfusion with the Ca2+-free KAsp external solution did not induce currents in HeLaCx43 cells that were not transfected with SpIH (0.4 ± 0.1 pA/pF, n = 5).
FIGURE 6. Depolarization induced cAMP uptake.
(A)
Im recorded from a HeLaCx43/SpIH cell during perfusion with Ca2+-free NaCl solution containing 500 μM cAMP. Initially the cell was held at Vh = 0 mV (not shown in the record). There was a weak (~1.3-fold) current increase over approximately 80 s when the cell was held at Vh = +50 mV. Further cell membrane depolarization to Vh = +80 mV at ~80 s significantly enhanced the SpIH current in a time dependent manner by ~threefold (when adjusted to the same Vh). (B) SpIH current recorded in a HeLaCx43/SpIH cell during perfusion with Ca2+-free KAsp solution. The SpIH current increase was associated with the time-dependent membrane current increase during depolarization (noted by a red circle), typical of hemichannel activity.
FIGURE 7. cAMP uptake via Cx26 hemichannels.
(A) Schematic of the experimental environment for HeLaCx26/SpIH cells. (B) Current recorded from a HeLa Cx26/SpIH-expressing cell. Perfusion with the Ca2+-free KAsp external solution induced a significant SpIH current increase over time. (C) Average of current densities measured from 5 cells: 6.4 ± 2.2 versus 32.3 ± 3.3 pA/pF, before and after perfusion, respectively; P = 0.008. 500 μM of cAMP was present in the external solution throughout the entirety of the experiment.
FIGURE 8. Single hemichannel currents.
(A) Current recorded from a HeLaCx26/SpIH cell. Perfusion with the Ca2+-free KAsp external solution did not induce a SpIH current increase in four preparations (5.9 ± 1.7 and 6.2 ± 1.9 pA/pF, before and after perfusion, respectively; P = 0.886). The operation of single functioning Cx26 hemichannels was visible throughout the experiment. Insert: segment of current recording on expanded time and current scales shows the opening and closing of a Cx26 hemichannel with a unitary conductance of ~280 pS. The external bath contained 500 μM cAMP. (B) Hemichannel currents elicited by biphasic 50 mV pulses recorded from HeLa Cx26 cells in a Ca2+-free KAsp solution. The all point current histograms yielded a conductance of ~290 pS.
FIGURE 9. Local hemichannel activation.
(A) Schematic illustrating an experiment with local application of cAMP. The perfusion pipette containing NaCl (*) or KAsp (**) solutions was brought close to the cell and a positive pressure was applied to allow the contents of the pipette to reach the cell membrane. Local application of cAMP via the external pipette did not affect the SpIH current in HeLaCx43/SpIH cells when the perfusion pipette contained the NaCl solution (*):6.6 ± 1.5 and 6.9 ± 1.3 pA/pF, before and during local cAMP application, respectively; n = 6, P = 0.456 (B). However, the SpIH currents did increase when the pipette contained the KAsp solution (**) (C). (D) Summary of current densities: 7.0 ± 1.6 and 23.9 ± 6.2 pA/pF, before and during local cAMP application, respectively; n = 6, P = 0.041. Currents marked in red in (B) and (C) correspond to recordings during the local application of cAMP; asterisks (*) and (**) indicate moments when a positive pressure was applied to the perfusion pipette.
FIGURE 10. Modulation of hemichannel activity.
(A) Schematic of the experiment. (B) SpIH current recordings from a HeLaCx43/SpIH cell perfused with Ca2+-free KAsp external solution containing 200 μM carbenoxolone. No SpIH current increase was detected during local application of cAMP (red current traces) in five cell preparations (6.8 ± 2.2 and 6.6 ± 2.8 pA/pF, before and during local application of cAMP, respectively; P = 1.0). (C) SpIH current recorded from connexin deficient HeLa parental cells transfected with SpIH during local application of cAMP (red current traces; 6.8 ± 1.9 and 8.7 ± 2.8 pA/pF, before and during local application of cAMP, respectively; n = 10, P = 0.734.
Al-Mehdi,
ATP-independent membrane depolarization with ischemia in the oxygen-ventilated isolated rat lung.
1998, Pubmed
Al-Mehdi,
ATP-independent membrane depolarization with ischemia in the oxygen-ventilated isolated rat lung.
1998,
Pubmed
Anselmi,
ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear.
2008,
Pubmed
Beahm,
Hemichannel and junctional properties of connexin 50.
2002,
Pubmed
Bruzzone,
A self-restricted CD38-connexin 43 cross-talk affects NAD+ and cyclic ADP-ribose metabolism and regulates intracellular calcium in 3T3 fibroblasts.
2001,
Pubmed
Bukauskas,
Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells.
2000,
Pubmed
Calabresi,
Sodium influx plays a major role in the membrane depolarization induced by oxygen and glucose deprivation in rat striatal spiny neurons.
1999,
Pubmed
Contreras,
Functioning of cx43 hemichannels demonstrated by single channel properties.
2003,
Pubmed
Contreras,
Gating and regulation of connexin 43 (Cx43) hemichannels.
2003,
Pubmed
Contreras,
Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture.
2002,
Pubmed
Dahl,
Pannexin: to gap or not to gap, is that a question?
2006,
Pubmed
DeVries,
Hemi-gap-junction channels in solitary horizontal cells of the catfish retina.
1992,
Pubmed
Drawnel,
The role of the paracrine/autocrine mediator endothelin-1 in regulation of cardiac contractility and growth.
2013,
Pubmed
Dreier,
The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease.
2011,
Pubmed
Duarte,
Contribution of the extracellular cAMP-adenosine pathway to dual coupling of β2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle.
2012,
Pubmed
Ebihara,
Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels.
1995,
Pubmed
Eskandari,
Inhibition of gap junction hemichannels by chloride channel blockers.
2002,
Pubmed
Fan,
Perforated patch recording with beta-escin.
1998,
Pubmed
Gauss,
Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.
1998,
Pubmed
,
Echinobase
González,
Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels.
2006,
Pubmed
Hofer,
Extracellular calcium and cAMP: second messengers as "third messengers"?
2007,
Pubmed
Kalvelyte,
Connexins and apoptotic transformation.
2003,
Pubmed
Kanaporis,
Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.
2008,
Pubmed
,
Echinobase
Kang,
Connexin 43 hemichannels are permeable to ATP.
2008,
Pubmed
Kléber,
Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts.
1983,
Pubmed
Kléber,
Mechanism and time course of S-T and T-Q segment changes during acute regional myocardial ischemia in the pig heart determined by extracellular and intracellular recordings.
1978,
Pubmed
Kuzhikandathil,
The extracellular cAMP-adenosine pathway regulates expression of renal D1 dopamine receptors in diabetic rats.
2011,
Pubmed
Laing,
The gap junction protein connexin43 is degraded via the ubiquitin proteasome pathway.
1995,
Pubmed
Maurer,
Cell pairs isolated from adult guinea pig and rat hearts: effects of [Ca2+]i on nexal membrane resistance.
1987,
Pubmed
Plotkin,
Transduction of cell survival signals by connexin-43 hemichannels.
2002,
Pubmed
Quist,
Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.
2000,
Pubmed
Segretain,
Regulation of connexin biosynthesis, assembly, gap junction formation, and removal.
2004,
Pubmed
Shin,
Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate.
2001,
Pubmed
,
Echinobase
Srinivas,
Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels.
2005,
Pubmed
Steffens,
Regulation of connexons composed of human connexin26 (hCx26) by temperature.
2008,
Pubmed
Stout,
Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.
2002,
Pubmed
Sánchez,
Differentially altered Ca2+ regulation and Ca2+ permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome.
2010,
Pubmed
Trexler,
Voltage gating and permeation in a gap junction hemichannel.
1996,
Pubmed
Trexler,
Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.
1999,
Pubmed
Valiunas,
Electrical properties of gap junction hemichannels identified in transfected HeLa cells.
2000,
Pubmed
Valiunas,
Formation of heterotypic gap junction channels by connexins 40 and 43.
2000,
Pubmed
Valiunas,
Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.
2002,
Pubmed
Valiunas,
Gap junction channels formed by coexpressed connexin40 and connexin43.
2001,
Pubmed
Valiunas,
Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.
2005,
Pubmed
Wang,
Paracrine signaling through plasma membrane hemichannels.
2013,
Pubmed
