KAUNAS UNIVERSITY OF MEDICINE
Mindaugas Račkauskas
GATING AND PERMEABILITY PROPERTIES OF GAP JUNCTION CHANNELS AND HEMICHANNELS FORMED
OF CONNEXINS: M30.2, 40, 43 AND 45
Doctoral Dissertation
Biomedical Sciences, Biophysics (02 B)
Kaunas, 2007
Dissertation has been prepared at the department of neuroscience of Albert Einstein College of Medicine of Yeshiva University (New York, USA) in 2004 - 2006.
Dissertation is defended extramurally.
Scientific supervisor:
Prof. Dr. Habil. Feliksas Bukauskas (Yeshiva University, Biomedical Sciences, Biophysics ⎯ 02 B).
CONTENTS
1. ABBREVATIONS...4
2. INTRODUCTION ...5
3. AIM AND OBJECTIVES...8
4. SCIENTIFIC VALUE ...9
5. LITERATURE REWIEW...10
5.1. Structure of connexins... 10
5.2. Connexons... 12
5.3. Gap junction channels ... 12
5.4. Gap junction clustering ... 14
5.5. Expression patterns of connexins in different tissues ... 16
5.6. Heterotypic compatibility among Cx40, Cx43 and Cx45 ... 17
5.7. Voltage gating and cell-to-cell signaling asymmetry of heterotypic junctions... 18
5.8. Hemichannels... 20
5.9. Dye permeability studies... 21
6. METHODS ...25
6.1. Cell Lines and culture conditions... 25
6.2. Solutions... 25
6.3. Electrophysiological Measurements ... 25
6.4. Fluorescence imaging and Dye transfer studies ... 26
6.5. Data and statistical analysis ... 28
7. RESULTS ...29
7.1. Fluorescence imaging of Cx40 and Cx43 heterologous cell pairs ... 29
7.2. Electrophysiological examination of Cx40/Cx43 ... 30
7.3. Compatibility between Cx43 and Cx45 ... 31
7.4. Compatibility between Cx40 and Cx45 ... 34
7.5. Compatibility between Cx40 and Cx43 ... 37
7.6. Unitary gating events of heterotypic gap junction channels ... 38
7.7. Permeability of homotypic GJ channels... 42
7.8. Permeability of heterotypic junctions ... 46
7.9. Dye uptake by Cx30.2 transfectants... 48
7.10. Mefloquine effect on hemichannels ... 52
7.11. Junctional conductance and voltage sensitivity, effect of mefloquine... 54
7.12. Dye uptake by HeLa cells and cardiomyocytes expressing hCx31.9-EGFP... 56
7.13. Electrophysiological measurements of hemichannel function... 57
8. RESULTS SUMMATION...59
9. CONCLUSIONS...71
10. ACKNOWLEDGEMENTS ...72
11. REFERENCES...73
1. ABBREVATIONS
Å - angstrom AF - Alexa Fluor 350 AV - atrioventricular
CFP - cyan fluorescence protein Cx – connexin
Da - unified atomic mass unit (u), or dalton
DAPI - 4’,6-diamidino-2-phenylindole dihydrochloride DTT – dithiothreitol (Cleland's reagent)
EGFP - enhanced green fluorescence protein EtdBr - ethidium bromide
ER – endoplasmic reticulum Gj - junctional conductance GJs - gap junctions
GSH - glutathione LY - Lucifer yellow PrI - propidium iodide SA -sinoatrial
TBCl - true blue chloride Vj - transjunctional voltage γ - single channel conductance
2. INTRODUCTION
Connexins (Cxs), a large family of homologous membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells (Harris, 2001; Paul, 1986). Each GJ channel is composed of two hemichannels (connexons), which in turn are composed of six Cx subunits. Thus far, at least twenty distinct Cx isoforms have been cloned (Sohl and Willecke, 2004). Cell-cell communication can be organized through homotypic (same Cx isotype in both hemichannels), heterotypic (hemichannels differ in Cx isotypes) or/and heteromeric (different Cx isotypes at least in one hemichannel) channels that vary highly in conductance, perm- selectivity and gating properties (Harris, 2001). Cx30.2, Cx40, Cx43 and Cx45 are expressed in different tissues, but most abundantly in cardiovascular and central nervous systems. Cx40, Cx43 and Cx45 have been regarded as the three major cardiac connexins (Sohl and Willecke, 2004;
Severs et al., 2004). MCx30.2 has been recently identified as the fourth cardiac connexin in mouse;
its putative human homologue is Cx31.9 (Kreuzberg et al., 2005). MCx30.2, which is expressed most abundantly in the conduction system of the heart (Kreuzberg et al., 2005), is also expressed in neurons of hippocampus and different nuclei of the cerebellum, thalamus and brainstem of adult mice (Willecke and Kreuzberg, 2006); hCx31.9 is the putative human ortholog of mCx30.2.
(Nielsen et al., 2002; White et al., 2002). Given the specific expression patterns of these connexins, their capacity to form heterotypic and heteromeric junctions will significantly influence nature of intercellular communication and the spread of excitation within and between different regions of the heart (Gros and Jongsma, 1996; Coppen et al., 1998; Verheijck et al., 2001; Kreuzberg et al., 2006).
Blood vessels express Cx40, Cx43, Cx45 along with Cx37, with Cx40 most abundantly expressed in endothelial cells and Cx43 in smooth muscle cells (Bruzzone et al., 1993). In the CNS, astrocytes abundantly express Cx43, endothelial cells of the blood-brain barrier express Cx40 and Cx43 (Nagasawa et al., 2006), and neurons of the olivocerebellar system express Cx45 (Van Der Giessen et al., 2006). Accordingly, a number of studies have reported malformation and dysfunction not only of the heart, but also of the whole cardiovascular system of mice in which Cx40, Cx43 or Cx45 were knocked out (Kirchhoff et al., 2000; Kruger et al., 2000; Liao et al., 2001; Verheule et al., 1999). Only Cx40 knockout mice survive during the postnatal period and they exhibit severe dysfunction of cardiovascular system, such as impaired SA-nodal function, slowed conduction velocity in the atria and the AV-nodal region, impaired conduction in bundle branches, structural abnormalities of endothelial and smooth muscle layers in the blood vessels, deficient conduction of vasodilator stimuli along the vessel wall, elevated mean arterial pressure and blood pressure regulation abnormalities (Kirchhoff et al., 1998; van Rijen et al., 2001; Bagwe et al., 2005; de Wit et al., 2006).
Several studies reported formation of functional heterotypic junctions between cells expressing Cx45 with those expressing Cx40 and Cx43 (Bukauskas et al., 2002; Cottrell and Burt, 2001; Elenes et al., 2001; Valiunas et al., 2002; Valiunas et al., 2000). Electrophysiological studies in Xenopus oocytes (Bruzzone et al., 1993) and HeLa transfectants (Haubrich et al., 1996) reported that Cx40 and Cx43 do not form functional heterotypic channels. Furthermore, in the HeLa transfectants, where cells expressing Cx43 were co-cultured with those expressing Cx40, immunohistochemical evidence for heterotypic JP formation was lacking (Haubrich et al., 1996) suggesting that Cx40 and Cx43 hemichannels are unable to dock or to cluster into visible junctional plaques (JPs). Those studies were stimulated by findings that Cx40 and Cx43 are expressed in different cell types in the cardiovascular system that come into direct contact. Cx43 is the major connexin expressed in working myocardium and smooth muscle cells of the blood vessels while Cx40 is mainly expressed in the atrium, atrioventricular node, AV bundle and endothelial cells.
These findings suggested that intercellular communication can be spatially regulated by the selective expression of different connexins and an inability to form heterotypic junctions may control the pattern of communication between cells (Bruzzone et al., 1993). Whether Cx40, which is expressed in the A-V bundle and its branches, and Cx43, which is the major Cx of working cardiomyocytes (Severs et al., 2004), are compatible to pair heterotypically is highly important in understanding the functional interaction between the conduction system and working myocardium cardiomyocytes. The inability of Cx40 and Cx43 to pair heterotypically is functionally relevant; the absence of coupling between the conductive system and the working myocardium can assure a high speed of excitation propagation in the A-V bundle, its branches and subendocardial layer of Purkinje network. Co-expression of Cx40 and Cx43 in the distal part of the conductive system (Gros and Jongsma, 1996), explains excitation transfer through homotypic Cx43 junctions at transitions formed at Purkinje-muscle (P-M) connections presumably through homotypic Cx43 junctions. For more than a decade, it was assumed that Cx40, Cx43, and Cx45 determine the propagation of excitation in the heart. Cx30.2 was recently characterized as a fourth cardiac Cx, which is expressed preferentially in the SA- and AV-nodal regions of the mouse heart (Kreuzberg et al. 2005).
Extent and pattern of Cx isoform expression together with cytoarchitecture and excitability of cells determine the velocity of excitation spread in different regions of the heart. MCx30.2 and Cx45 form low conductance GJ channels in the SA node. The working cardiomyocytes of atria and ventricles express mainly Cx40 and Cx43, which form GJ channels of high conductance that facilitate the fast conduction necessary for efficient mechanical contraction. Cx30.2 and Cx45 may determine higher intercellular resistance and slower conduction in the SA and AV nodal regions than in ventricular conduction system or the atrial and ventricular working myocardium. Several
studies reported formation of functional heterotypic junctions between cells expressing Cx40 and Cx43. We determined that Cx40 and Cx43 are not compatible to dock and to form heterotypic gap junction channels that is important in determining the spread of excitation in the conduction system of ventricles and excitation transfer between subendocardial Purkinje network and working cardiomyocytes.
Permeability of Cx-based hemichannels to metabolites is important to cell function under normal and pathological conditions in different organs and tissues. Among the multiple types of channels expressed in vertebrate cells, only gap junction channels are known to allow intercellular communication between the cytoplasms of adjacent cells in tissues and in some configurations these channels allow direct communication between the cytoplasm and the extracellular media. Various compounds up to a molecular mass of 1000 Da can be exchanged by passive diffusion through gap junctional conduits, i.e. metabolites, ions, second messengers, water and electrical impulses (Kumar et al., 1996). Also hemichannels can function under certain conditions, allowing cellular release and uptake of diffusible molecules.
3. AIM AND OBJECTIVES
AIM
To examine gating and permeability properties of homotypic and heterotypic gap junction (GJ) channels assembled between cells expressing cardiac connexins.
OBJECTIVES
1. To evaluate compatibility and gating properties of homotypic and heterotypic gap junction channels formed of mCx30.2, Cx40, Cx43, and Cx45 in HeLa cells.
2. To determine permeability properties of homotypic and heterotypic gap junction channels formed of mCx30.2, Cx40, Cx43 and Cx45 in HeLa cells.
3. To evaluate conductance and permeability of hemichannels formed of mCx30.2 in HeLa cells.
4. To evaluate permeability properties of hemichannels formed of hCx31.9 in transfected rat cardiac myocytes and HeLa cells.
4. SCIENTIFIC VALUE
Cx30.2, Cx40, Cx43 and Cx45, which are principal cardiac connexins forming gap junctions between cardiomyocytes of the conduction system and working myocardium of atria and ventricles.
Until now it was controversy in regards an ability Cx40 and Cx43 to form heterotypic gap junctions. Electrophysiological studies in Xenopus oocytes (Bruzzone et al., 1993) and HeLa cells (Gu, et al., 2000; Haubrich et al., 1996) reported that Cx40 and Cx43 do not form functional heterotypic channels. But, subsequent studies, performed in HeLa and RIN cell transfectants reported low levels of functional coupling attributed to Cx40/Cx43 heterotypic channels (Valiunas et al., 2000; Cottrell and Burt, 2001). Both studies concluded that Cx40/Cx43 channels can be functional despite reporting differences in properties such as Vj gating, single channel conductance and open channel current rectification. We think that an expression of Cx40 by cells forming the His bundle, its branches and terminal network of Purkinje fibers at early stages of development is important for their differentiation into conductive system independently from working cardiomyocites of ventricles expressing mainly Cx43. Given the potential importance of Cx40 and Cx43 compatibility in the cardiovascular system, the CNS, and other organs, we re-examined this issue, taking advantage of GFP-tagged connexins that allowed us to visualize JP formation and record electrophysiologically from the same cell pairs. Our experiments demonstrate that Cx40 and Cx43 are incompatible and that the failure to observe functional Cx40/Cx43 heterotypic channels results from an inability of the component hemichannels to dock and/or to cluster into JPs.
In dye permeability studies of Cx30.2, Cx40, Cx43 and Cx45, we used a novel technique, which allow us to increase dye detection sensitivity, in cases where coupling is weak or channel permeability is low. We used focused excitation light directed only at dye-recipient cell. The latter allowed us to avoid emission light scattering from dye donor cell as well from the dye-filled pipette which can obscure dye transfer to the recipient cell in cases where permeability is low or give the appearance of dye transfer when it is, in fact, absent.
MCx30.2 is most abundantly expressed in sinoatrial and atrioventricular nodal regions of the heart. Its putative human ortholog, hCx31.9, also form functional hemichannels, which like mCx30.2 cell-cell channels are permeable to cationic dyes up to ~400 Da in size. Unitary conductance of the fully open mCx30.2 hemichannel is ~20 pS, whichis approximately double that of the cell-cell channel. Cx30.2 is important in conduction system of the heart by slowing conduction and protecting ventricles from over excitation during atrial tachycardia.
5. LITERATURE REWIEW
5.1. Structure of connexins
The family of connexin (Cx) genes comprises 20 members in mouse and 21 genes in the human genome. MCx33 occur only in the mouse genome and hCx25, hCx59 only the human genome. The biological explanation is not determined. All rest genes are orthologous pairs.
Orthologous connexins may not necessarily be expressed in the same tissue or cell type, since hCx30.2, hCx31.9, hCx40.1 and hCx62 were recently found to be additionally expressed in the human heart, whereas their mouse orthologues mCx29, mCx30.2, mCx39 and mCx57 are not, as deduced from Northern blot results. Connexins are a protein family, which molecular mass range from 26 to 62 kDa. Cx are named by their molecular mass determined by amino acid sequence. For example Cx32 molecular mass is 32007 Da, Cx43-43036 Da (Aidley and Stanfield, 1996).
Connexins can be divided into subgroups (α, β or γ) with respect to their extent of sequence identity and length of the cytoplasmic loop (Willecke et al., 2002). α group with shorter N-termini (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32), β group (Cx33, Cx37, Cx38, Cx40, Cx42, Cx43, Cx45, Cx46, Cx50, Cx56, Cx57) and γ group - Cx36 (Dhein, 2002; Willecke et al., 2002; Rozental et al., 2000).
Gap junction channel is composed of two hemichannels (connexons). Each connexon composed of six connexin subunits in the plasma membrane and can dock to another connexon of an adjacent cell in the plasma membrane. Connexins have four alpha helical transmembrane domains (TM1 to TM4), intracellular N- and C-termini, two extracellular loops, and a cytoplasmic loop (CL) (Hertzberg et al., 1988; Yancey et al., 1989; Yeager et al., 1998). Fig. 1 shows topological model of a connexin.
Connexins have hydrophobic and hydrophilic regions. Both extracellular loops participate connecting hemichannels from adjacent cells and forming channel. The intracellular loop that connects the second and third transmembrane domains and the carboxy terminus have unique amino acid sequences that are responsible for the different molecular weights and specific channel properties of the connexins.
A
B
Fig. 1. Topological model of a connexin.
A. The transmembrane domains (M1– M4) are shown in white, the N-terminus and extracellular domains are shown in gray (hatched), and the intracellular hydrophilic loop connecting the second and third transmembrane domains (A) and the C-terminal tail (B) are shown in black.
B. Comparison of the sequences of multiple members of the connexin family. The amino acid number begins at the N- terminus. The gray (hatched) and white segments correspond to the regions of the molecules shown in A. Divergent sequences, shown in black, are located mainly in the intracellular loop (A) and the C-terminal tail (B) regions (adapted from (Kanno and Saffitz, 2001)).
5.2. Connexons
After iRNR translation, synthesized connexins appear in ER, and in Golgi complex oligomerise into connexons (van Veen et al., 2001). Each connexon is composed of six Cx subunits, and it is ~7.5 nm high and 7 nm in diameter. The pore inside is 1.5 up to 2 nm diameter and allow molecules up to 1 kDa, like metabolites, nucleotides, smaller saccharides, second messengers and others (Willecke et al., 2002), to be transferred from cell to cell. They may also transmit other regulatory molecules of unknown identity.
Other groups of researchers demonstrate smaller diameter of the pore around 1,5 nm and smaller molecular mass of substances (~ 1 kDa) to be transferred (Dhein, 2002; Aidley and Stanfield, 1996; Lampe and Lau, 2000).
5.3. Gap junction channels
Connexins form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between adjacent cells (Bennett, 1994; Elfgang et al., 1995; Goodenough et al., 1996; Paul, 1986). Channels formed by individual connexins exhibit distinct biophysical properties including unitary conductances, pH dependence, voltage dependence, and selective permeability to ions and small molecules such as fluorescent dyes. The permeability and gating characteristics of the channels are indeed dependent on the protein isoform and on posttranslational modifications.
Voltage sensitivity is likely to be particularly important in regulating intercellular coupling between excitable cells. Cx40 forms channels with larger conductances (150–160 pS) than Cx43 channels (Brink et al., 1996; Beblo et al., 1995; Bukauskas et al., 1995; Beblo and Veenstra, 1997). In contrast, Cx45 channels exhibit a much lower main state conductance of ~ 30 pS and are highly sensitive to transjunctional voltage (Veenstra, 1996; Veenstra et al., 1994). Cx43 channels have main conductance states of 90–115 pS and are relatively insensitive to changes in transjunctional voltage compared with channels composed of Cx45 (Moreno et al., 1994; Brink et al., 1996;
Veenstra, 1996; Moreno et al., 1992). Individual differentiated cells express more than a single species of connexin and the possibility exists for the formation of individual channels composed of more than one isoform. Combinations of connexins in hybrid channels are illustrated in Fig. 2.
Individual connexons can be homomeric or heteromeric, and individual GJ channels can be homotypic, heterotypic or heteromeric. The potential number of unique combinations of heteromeric connexons and heteromeric GJ channels is very large. Little is known about the natural occurrence or biological significance of hybrid channels in the heart or other differentiated tissues.
Some but not all homomeric connexons can form heterotypic channels.
Fig. 2. Nomenclature for connexons and gap junction channels formed by one or more connexins.
Individual connexons can be homomeric or heteromeric, and individual GJ channels can be homotypic, heterotypic or heteromeric. Modified from Kumar and Gilula (Kumar and Gilula, 1996).
Gap junctions are located in zones of close intercellular contact, the nexus region (or in the heart the intercalated disk) where gap junctional channels are organized in clusters. A gap junction channel pore is approximately 100–150 Å in length and 12.5 Å width, bridging the 20Å gap between the two neighboring cells consists of two hemichannels. Two connexons interact via H- bonds between the extracellular loops of their connexins. An individual intercellular channel is created by stable, noncovalent interactions of two hemichannels, referred to as connexons, located in the plasma membranes of adjacent cells. Fig. 3.
The process of docking and how the two connexons find each other is poorly understood. It is possible that no other factors are necessary for channel or plaque formation because purified connexons tend to aggregate in filaments and sheets (Stauffer et al., 1991). Formation of gap junctions requires appropriate cell adhesion, especially that mediated by Ca2+ - dependent molecules (cadherins) (Jongen et al., 1991).
Earlier studies in Xenopus oocytes and HeLa cells suggested that Cx40 cannot form functional heterotypic channels with Cx43 (Bruzzone et al., 1993; White et al., 1995; Elfgang et al., 1995; Haubrich et al., 1996), but more recent experiments involving transfected HeLa cells indicate that heteromeric Cx40/Cx43 channels do form and exhibit asymmetrical conductance properties (Valiunas et al., 2000; Cottrell and Burt, 2001). Recent evidence also suggests that Cx43 and Cx45 can form both heteromeric connexons and homomeric, heterotypic channels (Moreno et al., 1995;
Koval et al., 1995). These findings have potential implications for intercellular coupling in specific regions of the heart, such as the interface between the sinus node and atrial myocardium or Purkinje fibers and ventricular myocardium.
Fig. 3. Schematic of gap junction channels.
Each apposed cell contributes a hemi-channel to the complete gap junction channel. Each hemichannel is formed by six protein subunits, called connexins. Closure is achieved by connexin subunits sliding against each other and tilting at one end, thus rotating at the base in a clockwise direction. Gap junction channels allow small molecules (molecular weight:
<1000 Da or exceeding an ionic radius: 0.8–1.0 nm) to be transferred from cell to cell. The darker shading indicates the portion of the connexon embedded in the membrane (adapted from (Kandel et al., 1995)).
5.4. Gap junction clustering
Cxs cluster into plaques at locations of cell-cell contact. Coupling was always absent in the absence of plaques, even in the presence of small plaques. Plaques exceeding several hundred channels always conferred coupling, but only a small fraction of channels were functional.
These data indicate that clustering may be a requirement for opening of gap junction channels.
Ultra structurally, GJs have been identified as areas of close membrane apposition that, in freeze- cleaved replicas, can be seen to consist of tightly clustered particles, with each particle representing a GJ channel (McNutt and Weinstein, 1970; Goodenough and Revel, 1970). Although clustering of GJ channels into plaques is the primary basis for their ultrastructural identification, variations in GJ channel packing have been described and interpreted as representing pleiomorphisms (Fujisawa et al., 1976; Bennett and Goodenough, 1978). Recently, a fusion protein consisting of Cx43 with enhanced green fluorescent protein attached to its carboxyl terminus (Cx43–EGFP) was transfected into mammalian cells and was shown to be transported to the cell surface and assembled into functional GJs at locations of cell–cell contact (Fig. 4) (Jordan et al., 1999).
Cell
Cell
Cell
Cell
Cell
Cell
Fig. 4. Demonstrate GJ plaques between adjacent Hela cells.
Plaques are in green colour (white arrows) and internalized plaques in degradation process are inside the cells. (From Bukauskas)
5.5. Expression patterns of connexins in different tissues
Expression patterns of connexins in different tissues suggest that heterotypic and heteromeric channels likely form and contribute to electrical and metabolic communication between cells. Cx40, Cx43 and Cx45 are principal cardiac Cxs capable of forming GJs between cardiomyocytes of the conduction system and working myocardium of atria and ventricles. Cx45, along with the recently identified mCx30.2 GJ protein, are most abundantly expressed in the sinoatrial and atrioventricular nodes (Kreuzberg et al., 2005), Cx40 is expressed in the atria and the conduction system of ventricles, while Cx43 is a major Cx forming GJs between working cardiomyocytes (Lo, 2000).
These distinct expression patterns are important for synchronous excitation of cells inside atria and ventricles and for imparting a substantial AV delay that is important for effective blood circulation (Kreuzberg et al., 2006). Expression of Cx40, Cx43, Cx45 along with Cx37 has also been reported in blood vessels, with most abundant expression of Cx40 in endothelial cells and Cx43 in smooth muscle cells (Bruzzone et al., 1993).
Expression patterns of all cardiac Cxs are shown in Fig. 5. Cx43, the major Cx forming GJ channels between working cardiomyocytes of atria and ventricles, is also expressed in the network of Purkinje fibers forming the distal part of the ventricular conduction system (Beyer et al. 1989;
Gourdie et al. 1993; Coppen et al. 1999a). Cx40 colocalizes with Cx43 in the atrial working myocytes and is abundantly expressed in the His bundle, bundle branches, and subendocardial network of Purkinje fibers, that is, structures with high conduction velocities. Furthermore, Cx40 is expressed in the central region of the AV node (Kirchhoff et al. 1998; Coppen et al. 1999b). Cx45 is abundantly expressed in the SA node and in the AV node with its posterior extension. In the His bundle and branches, Cx40 and Cx45 expression overlaps, and at the transition from bundle branches to subendocardial network of Purkinje fibers, the expression of Cx45 decreases, whereas expression of Cx43 increases (reviewed by Lo 2000). Expression of Cx30.2 in mice is mainly restricted to the cardiac conduction system, with the most abundant expression in the SA node, in the atrial vestibule, and in the AV node with its posterior extension. Only low levels of Cx30.2 protein were found in the His bundle and bundle branches (Kreuzberg et al. 2005). Cx30.2 expression was largely colocalized with Cx45 expression throughout the cardiac conduction system.
An overlapping expression pattern of Cx30.2 and Cx40 was found only in part of the AV node, the His bundle, and the bundle branches.
Fig. 5. Schematic of the heart (modified from Netter 1993) illustrating the spread of excitation and Cxs expression patterns.
Boxed areas connected with different regions of the heart indicate conduction velocities and Cxs expressed. (Kreuzberg et al. 2006).
In the CNS, astrocytes most abundantly express Cx43, endothelial cells of blood-brain barrier express Cx40 and Cx43 (Nagasawa et al., 2006), and neurons of the olivocerebellar system express Cx45 (Van Der Giessen et al., 2006).
It is not well established whether incompatibility of Cxs isoforms to form heterotypic junctions resulted from evolutionary directed necessity to perform specific function. This property could affect not only electrical and metabolic communication, but also cells differentiation during development. Typically, most embryonic cells express one or several Cx isoforms and strong connexin-mediated cell-cell coupling tend to eliminate intercellular gradients of permeants, which include ions, metabolites, small peptides, oligonucleotides and small interfering RNA (siRNA) (Harris, 2001; Neijssen et al., 2005; Valiunas et al., 2005). Thus, in order for neighboring cell populations to develop independently it may be important to express connexin isoforms that are incompatible to form heterotypic junctions, thereby preventing electrical synchronization, transfer of signaling molecules or metabolic communication.
5.6. Heterotypic compatibility among Cx40, Cx43 and Cx45
Originally, electrophysiological studies in Xenopus oocytes (Bruzzone et al., 1993) reported that Cx40 and Cx43 do not form functional heterotypic channels. A similar conclusion was reached using HeLa transfectants in studies that showed a lack of dye transfer (Elfgang et al., 1995) and a
lack of electrical cell-cell coupling (Haubrich et al., 1996); in the latter study, there was also no evidence that Cx40/Cx43 JPs formed when stained with Cx-specific antibodies suggesting that Cx40 and Cx43 hemichannels do not dock or cluster into JPs This conclusion was also supported by Gu et al. (Gu, 2000). However, subsequent studies, performed in HeLa and RIN cell transfectants, and in an A7r5 cell line endogenously expressing Cx40 and Cx43, reported functional coupling in these cells attributed to Cx40/Cx43 heterotypic channels (Cottrell and Burt, 2001; Valiunas et al., 2000). Both studies concluded that Cx40/Cx43 channels can be functional despite conflicting data regarding the polarity of Vj-gating, single channel conductance and I-V rectification of open channels. Cotrell and Burt (Cottrell and Burt, 2001; Valiunas et al., 2000) commented that the differences regarding voltage gating properties could be due to short duration (~0.5 s) of the pulse used by Valiunas et al. (Valiunas et al., 2000) to characterize steady state gj-Vj dependence and stated that by using this protocol they may have characterized “fast inactivation while changes in slow inactivation were not assessed”. It may be that the differences among all these studies likely arise from expression of intrinsic Cxs in HeLa and Rin cells, as well as other Cx(s) in addition to Cx40 and Cx43 in A7r5 cells.
5.7. Voltage gating and cell-to-cell signaling asymmetry of heterotypic junctions
A property common to GJ channels formed of all Cxs is sensitivity of gj to Vj (Bennett et al., 1991; Bukauskas and Verselis, 2004; Harris and Bevans, 2001). A common feature reported for Vj
gating is that steady-state gj does not decline to zero with increasing Vj, but reaches a plateau or residual conductance that varies from ~5% to 30% of the maximum gj depending on the Cx isoform (Moreno et al., 1994). Single channel studies showed that residual gj is due to incomplete closure of the GJ channel by Vj (Moreno et al., 1994; Weingart and Bukauskas, 1993). Residual conductance has its own gating properties (Bukauskas et al., 2002), which is likely to be the result of a partially closed channel. This fast gating has been related to the carboxyl terminal. As the CT is reduced in size, the channel loses its fast gating, and slow gating governs closing of the channel (Moreno et al., 2002), apparently separating the gating mechanism. Gating to different levels via distinct fast and slow gating transitions led to the finding that there are two different types of Vj sensitive gates. One is termed the fast Vj gate, characterized by transitions between open and residual conductance states, and the other is termed the slow Vj or loop gate, characterized by transitions between open and fully-closed states (Bukauskas and Verselis, 2004) (see Fig. 6). Gating mechanism is similar in other connexins but can be mediated by other molecular regions, as is seen in Cx26 where the N terminus is responsible for fast gating and residual conductance (Verselis et al., 1994; Oh et al., 1999).
Fig. 6. Voltage gating in HeLaCx43 and Novikoff cell pairs.
Pooled data of normalized gj vs. Vj dependence measured in Novikoff (open circles) and HeLaCx43 (filled circles) cell pairs. The solid line is a fit of all the points to a Boltzmann relation. Squares filled with lines of different orientations indicate areas in which Vj gating is mediated predominantly through the slow gate (rising lines), the fast gate (declining lines) and both gates (crossing lines). (Adapted from (Bukauskas and Verselis, 2004)).
Gating in heterotypic junctions is typically asymmetric with respect to Vj=0 and the degree of asymmetry depends on the intrinsic gating properties of the component hemichannels. Cx45 homotypic junctions exhibit the highest Vj-gating sensitivity among all members of the connexin family and this property contributes to the high degree of Vj-gating asymmetry in all heterotypic junctions containing Cx45 on one side, such as mCx30.2/Cx45 (Kreuzberg et al., 2005), Cx31/Cx45 (Abrams et al., 2006), Cx43/Cx45 (Bukauskas et al., 2002) and Cx40/Cx45. In all cases, there is higher Vj-gating sensitivity when the Cx45 side is made relatively negative, which has been shown to result predominantly from closure of the slow gate of the Cx45 hemichannel (Bukauskas et al., 2002). The fast gate of Cx45 also closes on this polarity, but its voltage sensitivity is shifted to higher Vjs. The fast and slow Vj-sensitive gates of Cx43 GJs also close on relative negativity.
Differences in the unitary conductances of component hemichannels resulted to higher Vj-gating asymmetry in Cx43/Cx45 junctions than that predicted by simple connection of two hemichannels exhibiting equal unitary conductances. Most of the Vj applied across a Cx43/Cx45 junction falls across the Cx45 hemichannel, which has ~3.5-fold smaller conductance than the Cx43 hemichannel, resulting in increased and decreased Vj-gating sensitivities of Cx45 and Cx43 hemichannels, respectively (Bukauskas et al., 2002).
The strong gj-Vj gating asymmetry of Cx43/Cx45 (Bukauskas et al., 2002) and Cx31/Cx45 (Abrams et al., 2006) heterotypic junctions produces signal transfer asymmetry that can be increased or decreased by making the cell expressing Cx45 relatively more negative or positive,
respectively. Therefore, this cell-to-cell signaling asymmetry seems to be a common feature of heterotypic junctions containing a Cx45 hemichannel on one side. Such signaling asymmetry may be functionally relevant in the CNS where signal propagation in one direction is preferred; it was shown an expression of Cx45 in neurons of the olivocerebellar system, in spinal motor neurons and in the ganglion cell and inner nuclear layers of mouse retina (Van Der Giessen et al., 2006; Chang et al., 2000; Guldenagel et al., 2000).
5.8. Hemichannels
Gap junction channels are formed by the union of two hemichannels, from the adjacent cells, and connecting the cytoplasm of the cells, allowing a flux of small ions and molecules such as ATP, glucose, glutathione (GSH), cAMP, and IP3 (Harris, 2001). Thus, gap junction channels allow electrical coupling, metabolic cooperation, and coordination of cell activities. Hemichannels are assembled in the endoplasmic reticulum and Golgi or post-Golgi compartments and are trafficked to the surface membrane. Hemichannels have been demonstrated in many cell types by immunocytochemistry, freeze-fracture EM, electrophysiological recording, dye uptake measurement, and/or biotinylation of cell surface proteins (Saez et al., 2005). Cx43 is expressed in many cell types, including astrocytes, fibroblasts, cardiomyocytes, and dendritic cells (SAEZ et al., 2003) Cx43 expressed in HeLa cells forms hemichannels with a small open probability (Contreras et al., 2003).
Hemichannel openings are increased by low extracellular Ca2+ (4, 5), which enhances release of small molecules such as ATP, glutamate, prostaglandin E2, and NAD+ (Evans et al., 2006), and may participate in paracrine signaling.
From numerous reports during the last several years, it has become evident that permeability of Cx-based hemichannels to metabolites is important to cell function under normal and pathological conditions in different organs and tissues (Bennett et al., 2003; John et al., 1999).
Unapposed hemichannels are closed in order to prevent loss of cytoplasmic solutes and entry of extracellular ions. Biophysical properties of Cx43 (Kondo et al., 2000) and Cx45 hemichannels have already been examined (Contreras et al., 2003; Valiunas, 2002). Cells expressing Cx43-EGFP demonstrate more ethidium bromide uptake than parental cells. It suggests that cells with more Cx43-EGFP expressed have more functional hemichannels at their cell surface. The hemichannel blockers La3+ and β - glycyrrhetinic acid, reduced ethidium bromide uptake in Cx43-EGFP cells but not in parental cells. Depolarization induced channel activity in Cx43 and Cx43-EGFP cells but not in parental cells. The single channel conductance is twice that of Cx43-EGFP and Cx43 cell-cell channels, as predicted from series arrangement of two hemichannels in a cell-cell channel. The fast
gating transitions are seen in Cx43 cell-cell channels and Cx43 hemichannels, whereas only slow transitions are seen in Cx43-EGFP cell-cell channels and hemichannels. In experiments with EGFP- Cx43 junctional plaques were seen, but no functional cell-cell channels or voltage activated hemichannels detected (Contreras et al., 2003). Hemichannels differ from cell-cell channels in their low open probability, at least if the membrane associated fluorescence of Cx43-EGFP is entirely due to hemichannels in the surface membrane. Recent study of Cx43-EGFP hemichannels in HeLa cells demonstrate that DTT increased dye uptake in Cx43 or Cx43-EGFP cells, but not in parental cells or cells expressing EGFP-Cx43, which does not form functional hemichannels. An increase in dye uptake was inhibited by hemichannel blockers; DTT increased hemichannel opening at positive voltages in Cx43-EGFP cells, but not in parental cells; GSH, a poorly membrane permeant, physiological-reducing molecule, enhanced hemichannel activity only when it was applied intracellularly; and the DTT effect was not due to changes in distribution, abundance, or phosphorylation state (as assessed by electrophoretic mobility) of hemichannels or changes in intracellular pH (Retamal et al., 2007; Evans et al., 2006). Connexin 43 has nine cysteine residues, three in each of the two extracellular loops and three in the cytoplasmic C-terminal domain.
Oxidation/reduction of intracellular cysteines may be involved in the regulation of the opening of Cx43-EGFP hemichannels. Hemichannels of other connexins with cysteine residues in the C terminus may also prove to be sensitive to redox potential.
Both electrophysiological studies in Xenopus oocytes expressing Cx40 (Beahm and Hall, 2004) and failure of Cx40 expression by HeLa cells to increase dye uptake indicate that Cx40 hemichannels are closed under normal perfusion conditions. Similarly, Cx45 hemichannels are probably closed at normal Ca2+ levels. Hemichannels formed by at least two cardiac connexins, mCx30.2 and Cx43, can contribute to ionic and metabolic exchange across the plasma membrane or in paracrine cell-to-cell signaling by small molecules.
5.9. Dye permeability studies
Cardiomyocytes express several connexins, which can form homotypic, heterotypic, and possibly heteromeric GJ channels. The composition and expression patterns of these channels determine excitation spread as well as metabolic cell-cell communication throughout the heart.
Permeability properties of homotypic GJ channels formed of Cx40, Cx43, and Cx45 have been examined quite extensively (Goldberg et al., 2004; Eckert, 2006). Cell-to-cell dye transfer studies have been used to examine whether dyes of different net charge and size can permeate gap junction channels formed of different Cx isoforms. Techniques used most often to evaluate dye permeability include monitoring dye transfer after scrape-loading in a cell monolayer or injection of dye into a
single cell through a microelectrode and monitoring fluorescence recovery after photobleaching (FRAP). These techniques are relatively easy to use, but they do not provide a measure of specific or single-channel permeability without assessment of the number of functional channels mediating dye transfer between cells. There were several attempts to determine specific or single channel permeability by correlating cell-to-cell transfer of fluorescent dyes or radioactive compounds with GJ channel numbers estimated from electronmicroscopy (Weingart, 1974; Brink and Dewey, 1978;
Biegon et al., 1987); reviewed in (Verselis and Veenstra, 2000). More recently, Weber et al. (Weber et al., 2004) reported single GJ channel permeabilities of six connexin isoforms expressed in Xenopus oocytes for fluorescent Alexa probes that vary in size, but possess the same net charge. A similar approach was used to quantify dye flux through single Cx40, Cx43 and Cx45 GJ channels expressed in HeLa cells (Valiunas et al., 2002; Valiunas, 2002). Recently, Eckert (Eckert, 2006) reported single channel permeabilities for LY and calcein in mammalian cells expressing Cx43 and Cx46, and Ek-Vitorin et al. (Ek-Vitorin et al., 2006) showed that permeability of Cx43 channels can be efficiently modulated by phosphorylation.
Previous studies have documented that GJs are permeable to secondary messengers involved in cell signaling, such as Ca2+, and IP3 (Saez et al., 1989; Sanderson, 1995; Niessen et al., 2000).
Metabolites such as glutamate, glutathione, ADP and AMP show ~15-fold higher permeabilities through Cx43 channels, whereas adenosine is ~10- fold more permeable through Cx32 channels (Goldberg et al., 1999; Goldberg et al., 2004). The above-mentioned signaling molecules and metabolites are comparable in molecular mass and net charge to the most often used fluorescent dyes suggesting that data obtained by studying dye transfer would likely be informative for metabolic communication as well.
Recently, mCx30.2 was added to the list of cardiac Cxs and mCx30.2 GJs were shown to transfer positively and negatively charged dyes such as Neurobiotin, EtBr, DAPI and AF350, but not larger dyes such as LY or PrI (Kreuzberg et al., 2006). GJs between HEK cells expressing hCx31.9, the human ortholog of mCx30.2, were reported to lack transfer of LY as well (Nielsen et al., 2002).
These results suggest that mCx30.2 channels do not appear to be particularly selective on the basis of charge, but restrict passage of molecules with molecular mass >400 Da. There are only a few studies examining permeability of heterotypic junctions quantitatively. Cao et al. (Cao et al., 1998), by using cell pairs of Xenopus oocytes and HeLa expressing Cx32 and Cx26, showed that Cx32/Cx26 heterotypic channels have a permeability to Lucifer yellow that is intermediate between Cx32 and Cx26 homotypic GJ channels. More recent study reported single channel permeabilities of heterotypic junctions formed of Cx26, Cx32, Cx37 and Cx43 (Weber et al., 2004) and concluded that in heterotypic GJ channels “permeability characteristics are determined by the more restrictive parental homotypic channel”.
Several groups have made an attempt to evaluate the permeability of a single GJ channel, Pγ, by using different methodical approaches. Biegon et al. (Biegon et al., 1987) attempted to determine the diffusion coefficient of the Cx43 channel endogenously expressed in Novikoff cells by correlating LY transfer with GJ channel numbers estimated from electron microscopic studies.
Eckert (Eckert, 2006) used this value of the diffusion coefficient to evaluate Pγ and found that it equal to 37 × 10–15 cm3/s. Valiunas et al. (Valiunas et al., 2002) calculated the amount of dye flux through single Cx40 and Cx43 GJ channels. Using these data and assuming a constant dye concentration gradient over short time intervals, Pγ for LY is estimated to be ~2 and 0.4 × 10–15 cm3/s for Cx43 and Cx40, respectively. Similarly, in another study (Valiunas, 2002) Pγ,LY for Cx45 is estimated to be ~0.3 × 10–15 cm3/s. Weber et al. (Weber et al., 2004) determined the permeabilities of Alexa-type dyes in Xenopus oocytes for several connexin isoforms including Cx43 by using a compartmental model (Nitsche et al., 2004) and found Pγ,AF-350 = 180 × 10–15 cm3/s for Cx43. Recently, Eckert (Eckert, 2006) reported Pγ,LY of ~17 × 10–15 cm3/s in BICR/M1Rk cells (a rat mammary tumor cell line that endogenously Cx43 channels) and ~39 × 10–15 cm3/s in HeLa Cx43 transfectants; our Pγ,LY value for HeLaCx43-EGFP cells (24.6 × 10–15 cm3/s) falls in between these two estimates. For more on the issue of potential sources for differences in those Pγ measurements see the discussions in (Weber et al., 2004; Eckert, 2006).
Defining the properties that govern cell-to-cell movement of solutes is a necessary step in elucidating how gap junctions affect cell growth and physiology. Second messenger molecules and ions diffusion over gap junction channels appears to be paramount to coordinate the function of the body. Fluorescent dyes are commonly used to study permeable (gap) junctions, but only rarely have quantitative values for junctional dye permeability been determined.
A number of studies have illustrated the selectivity properties of homotypic gap junctions for monovalent ions, particularly Cx43, Cx40 and Cx45 (Beblo and Veenstra, 1997; Wang and Veenstra, 1997; Veenstra et al., 1995; Veenstra et al., 1994). Investigation of monovalent cation (Cs+, Rb+, K+, Na+, Li+ , TMA+, TEA+) and anion (glutamate- , acetate-, aspartate-, nitrate-, F-, Cl-, Br-) permeability and conductance ratios leads to the conclusion that the rCx40 channel is not a simple aqueous pore. Monovalent cations apparently permeate through the pore in a partially hydrated state with reduced mobilities determined by the effective ionic radius relative to the limiting pore radius of < 6.6 Å. The rCx40 channel appears to have a low anionic permeability which could be due to one or more fixed anionic sites within the pore. The mechanism of anion permeation remains to be definitively determined although all results suggest that formation of cation-anion complexes is probable. The differences in cation, anion, and molecular (mono-, tri-, and tetrasaccharide) permeabilities suggest that differences exist in charge composition and pore size for rCx40 and rCx43 channels (Beblo and Veenstra, 1997).
The high-molecular-weight cut-off for permeation has traditionally been utilized to demonstrate that cells can communicate via gap-junction channels: fluorescent dyes (e.g., Lucifer yellow) were injected into the cytoplasm of one cell of a cell cluster. If gap junctions were present, dye spreading into neighboring cells could be observed. Injection of different fluorescent molecules has also been used to estimate gap-junction channel size via their molecular cutoff at 800 Da or 1.4 nm - to 1.6-nm pore diameter (Simpson et al., 1977; Schwarzmann et al., 1981; Flagg Newton et al., 1979). There are significant selectivity differences between different connexin isoforms.
Quantitative data on gap-junction permselectivity, particularly single-channel permeabilities for larger molecules, which would be comparable between different connexin isoforms, are not widely available (Nicholson et al., 2000). Cao et al. have performed correlated measurements of dye transfer and gap-junctional conductance in connexin-transfected HeLa cells and Xenopus oocytes.
This was one of the first studies to relate permeability measures to channel number. DAPI (49,6- diamidino-2-phenylindole), is only visible after binding to DNA and might be considered inadequate as a diffusion tracer. The permeability was estimated by counting the number of cells that had received dye after a specified period of time. Counting cells depends on an arbitrary threshold of fluorescence intensity (visibility) and will be different for different tracer dyes. The cell count is also not linearly related to junctional permeability, and, therefore, arguably an inaccurate measure. In an extension of this work, Weber et al. used the experimental techniques established for Xenopus oocytes and applied diffusion modeling to obtain quantitative single-channel permeability data. Using this approach, they were able to provide quantitative values for the permeability of a number of connexins and a set of structurally similar dyes. A similar approach using mammalian cells was used by Valiunas et al. who used dye perfusion into isolated HeLa cell pairs to quantify dye flux through single Cx40 and Cx43 gap-junction channels. There are only a few studies that report quantitative permeability data for channels of defined connexin composition. In Novikoff hepatoma, it was possible to correlate the mean cell-cell permeance for Lucifer yellow with mean particle numbers from electron microscopy and to obtain an estimate for the diffusion coefficient within a gap-junction channel (Biegon et al., 1987). In a similar approach, (Cao et al., 1998) normalized junctional permeability to junctional conductance, which can be used as an estimator for channel number if the single channel conductivity is known. This would then allow calculation of the single-channel permeance, which is a quantity that could be directly attributed to connexins that constitute the channel pore (Nicholson et al., 2000; Cao et al., 1998; Nitsche et al., 2004; Biegon et al., 1987) only provide a single-channel diffusion constant that can be interpreted only when certain assumptions are made about pore diameter and length of a gap-junction channel. (Valiunas et al., 2002) were primarily interested in transfer of biologically active molecules, and their data is presented in terms of molecules per second.
6. METHODS
6.1. Cell Lines and culture conditions
Experiments were performed in HeLa cells (a human cervix carcinoma cell line, ATCC No.
CCL-2) transfected with cDNAs encoding wild type Cx30.2, Cx40 or Cx43 and Cx45 and their fusion forms with color variants of green fluorescent proteins (EGFP or CFP). Cells were grown in Dulbecco's medium supplemented with 10% FBS. All media, sera, and culture reagents were obtained from Life Technologies (GIBCO BRL). The transfection procedure has been described previously (Jordan et al., 1999; Kreuzberg et al., 2005). To study homotypic junctions, cells of one type were seeded at a density of ~ 104 cells /cm2 on 22x22 mm number 0 coverslips placed in culture dishes. To study heterotypic junctions, two types of cells expressing different connexins were mixed in equal quantities and seeded on coverslips. In all heterotypic combinations at least one type of cell expressed a Cx fused with GFP. All cell lines expressing wild type Cxs before co- culturing were pre-labeled with DAPI or DiI (Molecular Probes, Inc., CA). Isolated heterotypic cell pairs were selected by identifying those in which the two cells were expressing GFPs differing in color or in which one cell expressed GFP and other was labeled with DAPI or DiI. Isolated heterotypic pairs were selected for recording between 24 and 60 hours after plating.
6.2. Solutions
HeLa cells were grown in Dulbecco's medium supplemented with 10% FBS in incubator with 5% CO2 and 37 Co. All media and culture reagents were obtained from Life Technologies (GIBCO BRL). The chamber was perfused with a modified Krebs-Ringer solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 5 glucose, 2 pyruvate (pH = 7.4). In most of the experiments patch pipettes were filled with pipette solution containing (in mM): 10 Na Asparpate, 130 KCl, 0.26 CaCl2, 1 MgCl2, 3 MgATP, 5 HEPES (pH = 7.2), 2 EGTA ([Ca2+] =~5 x 10-8 M) and filtered throught 0.22 µm pores. Solutions were adjusted to pH = 7.2 by using KOH. Tyrode and pipette solutions were made the same day before experiments.
6.3. Electrophysiological Measurements
For simultaneous electrophysiological and fluorescence recording, cells grown onto coverslips were transferred to an experimental chamber (Bukauskas, 2001) mounted on the stage of Olympus IX70 inverted microscope (Olympus America, Melville NY) equipped with
phase-contrast optics and a fluorescence imaging system. The chamber was perfused with a modified Krebs-Ringer solution.
Junctional conductance, gj, was measured using the dual whole-cell patch clamp (Neyton and Trautmann, 1985; White et al., 1985). Briefly, each cell of a pair was voltage clamped independently with a separate patch clamp to the same holding potential (V1=V2). By stepping the voltage in one cell and keeping the other constant (V2), junctional current (Ij) is measured as a change in a current to applied transjunctional voltage (Vj=V2-V1) in the unstepped cell, I2. Thus, gj
is obtained by dividing the change in I2 by Vj. With low levels of coupling, unitary junctional currents can be recorded as discrete quantal changes in the unstepped cell that are accompanied by equal and opposite quantal changes in the stepped cell. Voltages and currents digitized using a MIO-16X A/D converter (National Instruments, Austin TX) and acquired and analyzed using custom made software designed by B. Trexler and V. Verselis.
In all examined co-cultures of cells expressing different Cx isoforms at least one type of cells expressed Cxs fused with GFP. All cell lines expressing wild type Cxs before coculturing were pre- labeled with DAPI or DiI (Invitrogen Corp., Carlsbad, California). Isolated heterotypic pairs were selected by identifying pairs of cells in which cells were expressing GFPs of different color or one express GFP and other is labeled with DAPI or DiI. Isolated heterotypic pairs were selected for recording between 24 and 60 hours after their platting.
6.4. Fluorescence imaging and Dye transfer studies
Fluorescence signals were acquired using a Hamamatsu cooled digital camera mounted on an Olympus IX70 microscope and UltraVIEW software for image acquisition and analysis (Perkin Elmer Life Sciences, Boston, MA). Appropriate excitation and emission filters (Chroma Technology Corp., Brattleboro, VT) were used to image CFP, EGFP, LY, Alexa Fluor-350, ethidium bromide (EtBr), DAPI, PrIod and DiI. For dye transfer studies the experiments were performed in pre-selected cell pairs. Given dye was introduced into one cell of a pair (the donor cell) through a patch pipette in whole cell voltage clamp recording conditions. To minimize dye bleaching, the transfer of dye to the recipient cell was examined using time-lapse fluorescence imaging with exposure time of 0.5 s to excitation light applied every 5 s. Typically, establishment of a whole-cell recording in the donor cell with a dye-filled pipette showed rapid loading of the cell followed by dye transfer to the neighboring (recipient) cell. After allowing for dye spread for 6- 10 min, a whole cell recording was established in the recipient cell to measure gj. Total junctional permeability, Pdye, can be described by the equation,
)) (
(
C vol2x P
2 1
2 dye
∆ t C − C
= ∆
, (1)where C1 and C2 are the dye concentrations in cell-1 and cell-2, t is time,vol2 denotes volume of cell-2 (Hille, 2001; Verselis et al., 1986) at Vj=0 mV. Cell volume was approximated as a hemisphere. The diameter of a hemisphere was determined by averaging the longest and the shortest diameters of the cell; in average the volume of examined HeLa cells was ~1800 µm3. Assuming that dye concentration is proportional to fluorescence intensity (FI), which is supported by several studies and for different dyes and for concentrations of 1 mM or less (Valiunas et al., 2002; Ek-Vitorin and Burt, 2005), then C1 = k × FI1 and C2 = k × FI2, where k is a proportionality constant. We can describe single channel permeability, Pγ, as follows:
) )(
( 1, 2,
2 2
,
γ
γ
j n n
dye g
FI FI
t
FI x P vol
−
∆
= ∆ , (2)
where ∆FΙ2=(FΙ2,n+1−FΙ2,n) is the change in FI in cell-2 over the time, ∆t=(tn+1-tn); n is nth time point in the recording; (FΙ1,n−FΙ2,n) is the FI gradient between cells, and gj/γ is the number of open channels.
To increase sensitivity of dye transfer that is important in studies of cell pairs expressing low levels of coupling, we combined two methods to examine dye transfer. In the conventional method, the entire visible field is exposed to excited light so that light scattering from dye-donor cell as well as from the dye-filled pipette can obscure dye transfer to the recipient cell in cases where permeability is low and/or can give the appearance of dye transfer when it is, in fact, absent. To avoid these problems, in parallel with the conventional method, we used a focused light source, which periodically excited only dye-recipient cell. To minimize dye bleaching, imaging was performed in time-lapse mode, i.e., cells were periodically exposed (every 6 s or more) to a low- intensity light for ~0.4 s. We also used low dye concentrations to load the cells, typically 0.1 mM and below, which minimized photo toxicity, but still provided satisfactory signal intensities. We found no difference in fluorescence intensity over time when images were acquired once every 20 s or every 6 s, indicative that dye bleaching was minimal.
6.5. Data and statistical analysis
The data are expressed as the representative result or as the mean of at least three experiments
± SD. Means were compared using t-test. Values were considered significantly different when P<0.05. Data and statistical analysis were performed with SigmaPlot 2000 software. Voltage and current recorded data were analyzed with VTrex Technologies software.
7. RESULTS
7.1. Fluorescence imaging of Cx40 and Cx43 heterologous cell pairs
We examined whether there was evidence of Cx40/Cx43 heterotypic junction formation using several different types of heterologous cell pairings. In all heterologous pairing combinations examined, at least one cell type expressed a GFP-tagged form to allow visualization of JPs.
Cx43-CFP
Cx40-wt
Cx43-CFP Cx40-CFP
Cx43-EGFP
10 µm
Cx40-CFP
Cx43-EGFP
A B
E D
C
Cx40-CFP Cx43-EGFP
Cx45-CFP
Cx43-EGFP
F
15 µm Cx43-CFP
Cx40-wt
Cx43-CFP Cx40-CFP
Cx43-EGFP
10 µm
Cx40-CFP
Cx43-EGFP
A B
E D
C
Cx40-CFP Cx43-EGFP
Cx45-CFP
Cx43-EGFP
F
15 µm
Fig. 7. Absence of Cx40/Cx43 junctional plaque formation in HeLa cells.
(A) A cluster of HeLaCx40-CFP (cyan) and HeLaCx43-EGFP (red) cells exhibiting large junctional plaques formed of Cx40-CFP and Cx43-EGFP homotypic junctions; all in pseudocolors. In C is shown the inset from image (A) at lower fluorescence intensity to better visualize the junctional plaques (see arrows). (B) A cluster of two HeLaCx43-CFP cells (cyan) and a HeLaCx40-wt cell labeled with DAPI (red). (D-E) Fluorescence images in negative conversion from the region of interest indicated by the square in (A). Images of Cx40-CFP (D) and Cx43-CFP (E) from the same region of interest illustrate that small junctional plaques (shown by arrows) form but do not overlap. (F) Color overlap of cell pair formed of HelaCx43-EGFP (red) and HeLaCx45-CFP (cyan) cells. The arrow points to the junctional plaque.
Fig. 7A illustrates a group of HeLa cells expressing either Cx40-CFP (shown in cyan) or Cx43-EGFP (shown in red). The images show that homotypic JPs readily form, evident as large CFP or EGFP fluorescing puncta in regions of contacting Cx40-CFP and Cx43-EGFP cells, respectively. Typically, GFP fluorescence associated with JPs is significantly stronger than the fluorescence of connexin protein residing intracellularly or in the non-junctional membrane. This intense fluorescence of JPs is evident in Fig. 7C where the image from a region of interest (indicated by square in Fig. 7A) containing JPs is shown at substantially reduced intensity of
emitted light (see white arrows). No such large JPs were evident between contacting Cx40-CFP and Cx43-EGFP cells. Fig. 7B shows a Cx40 cell labeled with DAPI (shown in red) in contact with two Cx43-CFP cells. Neither of the Cx43-CFP cells shows clearly identifiable JPs with the contacting Cx40 cell. All together, we performed 35 experiments with fluorescence imaging and in each experiment we examined more than 20 heterologous cell pairs. We did not observe a single junctional plaque that showed overlap of Cx40-CFP and Cx43-EGFP fluorescence in > 700 cell pairs.
Upon closer inspection of these same images in white-black conversion to improve contrast (Fig. 7, D and E), small fluorescent puncta were occasionally visible at regions of contact between Cx40- and Cx43-expressing HeLa cells (white arrows in Fig. 7 D and E). We ascribe these small fluorescent puncta to JPs because when imaged over time, they like clearly identifiable JPs, where fixed in their movements to the plasma membrane visible by the distribution of labeled hemichannels (connexons) as a fluorescence line, whereas small fluorescent puncta typically representing cytosolic vesicles move randomly and independently of the movement of the plasma membrane. Images D and E are from the same region of interest but for CFP and EGFP fluorescence, respectively. The junctional puncta contain either CFP or EGFP fluorescence, but not both. Thus, these puncta cannot be Cx40-CFP/Cx43-EGFP heterotypic JPs, which would show co- localization of CFP and EGFP, and most probably represent Cx40-CFP/Cx45 and Cx43- EGFP/Cx45 heterotypic junctions as low levels of Cx45 expression in HeLa parental cells has been reported immunohistochemically (Butterweck et al., 1994). Double immunogold labeling of HeLa parental cells and HeLa transfectants expressing Cx40 and Cx43 also shows dispersed expression of GJ channel like particles formed of Cx45 (Hulser et al., 1997). This finding correlates with electrophysiological data showing that among 29 HeLa parental cell pairs only three were coupled with an average gj of 0.04 nS (Valiunas et al., 2000).
Fig. 7 F, which is pseudocolor overlap of EGFP (red) and CFP (cyan) signals, shows a junctional plaque in the region of contact between cells expressing Cx43-EGFP and Cx45-CFP.
This cell pair exhibited electrical coupling with gj=17 nS. Similarly, we have observed junctional plaques between cells expressing Cx40 and Cx45.
7.2. Electrophysiological examination of Cx40/Cx43
In electrophysiological examination of heterologous Cx40/Cx43 cell pairs, we found that a majority were coupled, but that coupling was typically weak with mean gj values for heterologous Cx40-CFP/Cx43-EGFP and Cx40/Cx43-EGFP combinations of 0.95+/-0.59 nS (n=25) and
1.06±0.81 nS (n=10), respectively. Only three cell pairs lacked coupling. All functionally coupled cell pairs exhibited gating asymmetry, which varied substantially among cell pairs. Examination of 35 homologous cell pairs formed of HeLa parental (untransfected) cells showed measurable coupling in only 5 cases, with gj values in each case below 0.1 nS. When HeLa parental cells were paired with HeLa cells expressing either Cx40-CFP or Cx43-EGFP, all cell pairs were coupled, but gj was low, approximately 0.3 and 0.5 nS, respectively. Thus, gj measured between cells expressing Cx40 and Cx43 is close to the sum of gjs measured in Cx43/Cx45(endogenous) are Cx40/Cx45 (endogenous) junctions, suggesting that they form in independent fashion. The data obtained from a number of different pairing configurations is summarized in Table 7.2.1. These results indicate that coupling between HeLa parental cells, which endogenously express Cx45, is low likely because of low levels of Cx45 expression and consequently inefficient JP formation. Coupling and junction formation involving Cx45 remains low but increases such that coupling levels of ~0.5 nS are achieved when at least one cell abundantly expresses Cx40 or Cx43.
Table 7.2.1. Averaged gjs in different homo- and heterologous cell pairs
Junctions
Number of cell pairs Conductance (gj), nS
Cx40/Cx40 21 27.8±7.1
Cx40/Cx40-CFP 36 23±5.2
Cx40-CFP/Cx40-CFP 103 20.2±2. 9
Cx40/Cx43-EGFP 10 1.06±0.81
Cx40-CFP/Cx43-EGFP 25 0.96±0.59
Cx40-CFP/Cx45 28 4.5±3.1
Cx43-EGFP/Cx43-EGFP 45 28±4
Cx43-EGFP/Cx45 35 13.3±3.5
Cx40-CFP/HeLa parental 6 0.33±0.27
Cx43-EGFP/HeLa parental 15 0.51±0.25
HeLa parental/HeLa parental 35 Only in 5 gj>0
7.3. Compatibility between Cx43 and Cx45
To examine compatibility between Cx43 and Cx45, we co-cultured HeLa cells expressing Cx43-EGFP with those expressing Cx45 and found that JPs readily formed between heterologous cells, consistent with previous reports (Bukauskas et al., 2002; Elenes et al., 2001). We selected and examined the electrophysiological properties of heterologous cell pairs that contained at least one clearly identifiable and sizeable JP formed of Cx43-EGFP/Cx45 heterotypic junctions.
A
B
C
Fig. 8. Voltage gating of Cx43/Cx45 heterotypic junctions.
(A) Summary of Gj-Vj dependence of Cx45/Cx43-EGFP junctions; the data are shown as open circles and as a solid line were obtained by applying Vj steps and ramps, respectively, to one cell of a cell pair. The increase in Gj at an expanded scale is shown in the inset; dashed lines show confidential interval at p=95 %. (B) Record of a junctional current (Ij) trace in response to voltage ramps and steps (upper trace) applied to the HeLaCx43-EGFP cell of a HeLaCx43- EGFP/Cx45 cell pair. Junctional conductance (gj) was calculated as Ij/Vj. (C) Ij-Vj relationship measured by applying Vj
ramps at different time intervals of the record (indicated by the horizontal segments labeled by letters, a-e; on Ij-Vj plot the squares correspond to segments shown on the Vj trace). Open squares were recorded when holding potentials in both cells were equal, i.e., Vj= 0 mV. Open circles were recorded during 100-160 s time period when voltage ramps were superimposed into the Vj steps.
