Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
J Gen Physiol
2008 Apr 01;1314:293-305. doi: 10.1085/jgp.200709934.
Show Gene links
Show Anatomy links
Gap junction channels exhibit connexin-specific permeability to cyclic nucleotides.
Kanaporis G
,
Mese G
,
Valiuniene L
,
White TW
,
Brink PR
,
Valiunas V
.
???displayArticle.abstract???
Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.
???displayArticle.pubmedLink???
18378798
???displayArticle.pmcLink???PMC2279171 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Properties of SpIH channels. (A) Voltage protocol (Vm) and whole cell currents (Im) recorded in control, untransfected (top), and SpIH-transfected HeLa cells in the absence (middle) or presence (bottom) of 50 μM cAMP. Im were elicited by 2-s voltage pulses delivered from a holding potential of 0 mV to test potentials between −20 and −120 mV, in 20-mV increments, and returning to a tail potential of +50 mV. Im increased in SpIH-transfected cells when cAMP was present in the pipette solution. (B) Average of tail current densities measured after voltage step to Vm = −100 mV from control cells (0.3 ± 0.05 pA/pF, n = 7), SpIH-transfected cells (5.3 ± 0.7 pA/pF, n = 13), and SpIH-transfected cells with 50 μM cAMP in the pipette (28.6 ± 3.1 pA/pF, n = 13). Data represent means ± SEM. (C) Dependence of the SpIH current on intracellular cAMP concentration. Solid line represents best fit of data to the Hill equation with EC50 = 7.1 μM, Hill slope = 2.01. Data represent means ± SEM, obtained from a total 48 cells at Vm = −100 mV. The experiments were performed in the presence of IBMX and 2’,5′ dideoxyadenosine.
Figure 2. Detection of intercellular transfer of cAMP. (A) Cell pair where one cell expressed SpIH-eGFP (green cell) and the other expressed RFP only (red cell). 500 μM cAMP was delivered via patch pipette into the red cell that did not express SpIH and SpIH currents were recorded from cell expressing SpIH in perforated patch mode. Both cells express Cx43. (B) Current recorded from the SpIH-expressing cell, in response to voltage pulses from a holding potential of 0 to −100 mV, returning to a tail potential of +50 mV. The arrow indicates the opening of the patch into whole cell recording mode and the beginning of cAMP delivery into the donor cell. SpIH current over time increased to steady state. The red bar on time axis indicates SpIH current activation time interval t during which current reaches saturation.(C) Segments of SpIH current records when the whole cell patch was opened (*) and 135 s later with saturated current (**).
Figure 3. (A and B) Plots of normalized tail current versus time recorded from the recipient cells after cAMP injection into the source cell for cell pairs expressing Cx43 (•): gj = 19 nS (A), and 2 nS (B); Cx26 (▾): gj = 25 nS (A), and 8 nS (B); and Cx40(○): gj = 22 nS (A) and 10 nS (B). SpIH activation time, t corresponds to the time when SpIH current reaches saturation in the recipient cell. The first order regression over the linear part of the plots (dashed line shown in Cx40 plot, A) was used to determine current activation rate ΔI/Δt. (C) Summarized plots of the SpIH tail current activation rate (ΔI/Δt) versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40(○). The solid lines correspond to the first order regressions with the following slopes: (30.0 ± 2.5) · 10−4 s−1/nS for Cx43, (7.3 ± 0.6) · 10−4 s−1/nS for Cx26, and (4.8 ± 1.1) · 10−4 s−1/nS for Cx40. The dashed lines are the 95% confidence intervals. The experiments were performed in the presence of IBMX and 2’,5′ dideoxyadenosine. Insert: current activation rate (ΔI/Δt) versus channel number for all three connexins with slopes of (1.6 ± 0.1) · 10−4 s−1/channel for Cx43 (•), (0.8 ± 0.07) · 10−4 s−1/channel for Cx26 (▾), and (0.6 ± 0.1) · 10−4 s−1/channel for Cx40 (○).
Figure 4. Plots of the SpIH activation time versus junctional conductance for Cx43 (•), Cx26 (▾), and Cx40 (○) in the absence (A) and presence (B) of phosphodiesterase (IBMX) and adenylate cyclase inhibitors (2’,5′ dideoxyadenosine). The SpIH activation times were reduced in IBMX and 2’5′dideoxyadenosine-treated cells and were reciprocally proportional to junctional conductance. The solid lines correspond to the first order regression and the dashed lines are the 95% confidence intervals. The arrows pointing to 15 and 20 nS indicate the threshold for detection of cAMP in recipient cell for Cx26 and Cx40, respectively.
Figure 5. (A–C) Plots of junctional conductance versus time from Cx43 (•, n = 9), Cx40 (○, n = 12), and Cx26 (▾, n = 12) expressing cell pairs, measured during the application of cAMP to the donor cell. Data represent means ± SEM. (D) Current recorded from the single SpIH-expressing cell with 20 μM cAMP in the pipette in perforated patch mode. There was no detectable SpIH current increase in perforated patch mode, and SpIH current increased dramatically when perforated patch was converted to the conventional whole cell patch clamp (around 15 min time mark). See text for details. All measurements were done in the presence of IBMX and 2’5′dideoxyadenosine.
Figure 6. Summary of LY flux data versus junctional conductance for Cx43, Cx26, and Cx40 cell pairs. Each data point for Cx26 (â–¾) represents a recipient cell fluorescence intensity over injected cell fluorescence intensity 12 min after LY injection (Valiunas et al., 2002). The solid lines correspond to the first order regressions and the dashed lines represent confidence intervals (95%) for each plot. See text for references on derivation of regression lines for Cx40 and Cx43.
Figure 7. Single channel properties of Cx26 channels. Voltage protocol (top) and single channel currents recorded from homotypic Cx26 cell pairs with pipette solutions of different ionic compositions. Top: K+ aspartate− pipette solution. Transjuctional voltage Vj
= 110 mV, the current histograms yielded γj of 111pS/109 pS for negative/positive Vj. Bottom: Na+ aspartate− pipette solution, Vj
= 90 mV, γj = 83 pS/85 pS for negative/positive Vj.
Figure 8. Selectivity properties of Cx43, Cx40, and Cx26 to various solutes relative to K+. The bars represent Na+/K+, cAMP/K+, and LY/ K+ ratios plotted on log scale for Cx43 (white bar), Cx26 (gray bar), and Cx40 (black bar).
Figure 9. Levitt model plotting D(x)/Do versus pore diameter for cAMP (○) and LY (•), where D(x) is the diffusion coefficient for a solute within the channel and Do is the equivalent within the cytoplasm. D(x)/Do is assumed to be approximated by the calculated flux ratios for cAMP/K+ and LY/K+. The continuous and dashed lines represent cAMP/K and LY/K+ ratios for Cx43 and Cx40, respectively.
Arnould,
Effect of aescine on hypoxia-induced activation of human endothelial cells.
1996, Pubmed
Arnould,
Effect of aescine on hypoxia-induced activation of human endothelial cells.
1996,
Pubmed
Banach,
The influence of surface charges on the conductance of the human connexin37 gap junction channel.
2000,
Pubmed
Barbe,
Cell-cell communication beyond connexins: the pannexin channels.
2006,
Pubmed
Barr,
Electrical transmission at the nexus between smooth muscle cells.
1968,
Pubmed
Beblo,
Monovalent cation permeation through the connexin40 gap junction channel. Cs, Rb, K, Na, Li, TEA, TMA, TBA, and effects of anions Br, Cl, F, acetate, aspartate, glutamate, and NO3.
1997,
Pubmed
Bedner,
A method to determine the relative cAMP permeability of connexin channels.
2003,
Pubmed
Bedner,
Selective permeability of different connexin channels to the second messenger cyclic AMP.
2006,
Pubmed
Bennett,
Gap junctions, electrotonic coupling, and intercellular communication.
1978,
Pubmed
Beyer,
Gap junctions.
1993,
Pubmed
Brink,
Gap junction channel gating and permselectivity: their roles in co-ordinated tissue function.
1996,
Pubmed
Brink,
Human connexin 43 gap junction channel gating: evidence for mode shifts and/or heterogeneity.
1996,
Pubmed
Brink,
Experimental diabetes alters connexin43 derived gap junction permeability in short-term cultures of rat corporeal vascular smooth muscle cells.
2006,
Pubmed
Bruzzone,
Pannexins, a family of gap junction proteins expressed in brain.
2003,
Pubmed
Caverzasio,
Characteristics and regulation of Pi transport in osteogenic cells for bone metabolism.
1996,
Pubmed
Chen,
Cyclic AMP diffusion coefficient in frog olfactory cilia.
1999,
Pubmed
Christ,
The role of gap junctions and ion channels in the modulation of electrical and chemical signals in human corpus cavernosum smooth muscle.
1993,
Pubmed
Christ,
Analysis of the presence and physiological relevance of subconducting states of Connexin43-derived gap junction channels in cultured human corporal vascular smooth muscle cells.
1999,
Pubmed
Christ,
Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone.
1996,
Pubmed
De Mello,
Impaired cell communication in the diabetic heart. The role of the renin angiotensin system.
2007,
Pubmed
DiFrancesco,
Serious workings of the funny current.
2006,
Pubmed
Elfgang,
Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.
1995,
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
Gemel,
Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells.
2004,
Pubmed
Goldberg,
Selective permeability of gap junction channels.
2004,
Pubmed
Gollob,
Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation.
2006,
Pubmed
Harris,
Connexin channel permeability to cytoplasmic molecules.
2007,
Pubmed
Hernandez,
Unitary permeability of gap junction channels to second messengers measured by FRET microscopy.
2007,
Pubmed
Iizuka,
Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle.
1994,
Pubmed
Kobayashi,
Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling.
1989,
Pubmed
Levitt,
General continuum theory for multiion channel. II. Application to acetylcholine channel.
1991,
Pubmed
Levitt,
General continuum theory for multiion channel. I. Theory.
1991,
Pubmed
Mathias,
Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.
1990,
Pubmed
Melman,
Pathophysiology of Erectile Dysfunction.
1999,
Pubmed
Meşe,
Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss.
2004,
Pubmed
Niessen,
Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate.
2000,
Pubmed
Oliva,
Calculation of time constants for intracellular diffusion in whole cell patch clamp configuration.
1988,
Pubmed
Paznekas,
Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia.
2003,
Pubmed
Ponsioen,
Direct measurement of cyclic AMP diffusion and signaling through connexin43 gap junctional channels.
2007,
Pubmed
Rackauskas,
Permeability of homotypic and heterotypic gap junction channels formed of cardiac connexins mCx30.2, Cx40, Cx43, and Cx45.
2007,
Pubmed
Ramanan,
Non-stationary fluctuation analysis of macroscopic gap junction channel records.
2005,
Pubmed
Shin,
Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate.
2001,
Pubmed
,
Echinobase
Suchyna,
Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.
1999,
Pubmed
Tansey,
Ca(2+)-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells.
1994,
Pubmed
Tsien,
Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut end method.
1976,
Pubmed
Valiunas,
Electrophysiological properties of gap junction channels in hepatocytes isolated from connexin32-deficient and wild-type mice.
1999,
Pubmed
Valiunas,
Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells.
1997,
Pubmed
Valiunas,
Formation of heterotypic gap junction channels by connexins 40 and 43.
2000,
Pubmed
Valiunas,
Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions.
2005,
Pubmed
Valiunas,
Cardiac gap junction channels show quantitative differences in selectivity.
2002,
Pubmed
Valiunas,
Gap junction channels formed by coexpressed connexin40 and connexin43.
2001,
Pubmed
Valiunas,
Biophysical characterization of zebrafish connexin35 hemichannels.
2004,
Pubmed
van Rijen,
Human connexin40 gap junction channels are modulated by cAMP.
2000,
Pubmed
van Veen,
Cardiac gap junction channels: modulation of expression and channel properties.
2001,
Pubmed
Veenstra,
Selectivity of connexin-specific gap junctions does not correlate with channel conductance.
1995,
Pubmed
Wang,
Monovalent ion selectivity sequences of the rat connexin43 gap junction channel.
1997,
Pubmed
Weidmann,
The diffusion of radiopotassium across intercalated disks of mammalian cardiac muscle.
1966,
Pubmed
Weidmann,
Electrical constants of trabecular muscle from mammalian heart.
1970,
Pubmed
Yum,
Human connexin26 and connexin30 form functional heteromeric and heterotypic channels.
2007,
Pubmed
Zhao,
Connexin26 is responsible for anionic molecule permeability in the cochlea for intercellular signalling and metabolic communications.
2005,
Pubmed
