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. 2013 Jun 6:4:75.
doi: 10.3389/fphar.2013.00075. eCollection 2013.

Cyclic nucleotide permeability through unopposed connexin hemichannels

Virginijus Valiunas  1
Affiliations

Cyclic nucleotide permeability through unopposed connexin hemichannels

Virginijus Valiunas. Front Pharmacol. .

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 (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.

Keywords: connexin26; connexin43; cyclic AMP; electrophysiology; gap junction; permeability.

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Figures

FIGURE 1
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
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
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
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
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
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
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
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
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
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.
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