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. 2011 Jan 18;11:6. doi: 10.1673/031.011.0106

Characterization of Nonjunctional Hemichannels in Caterpillar Cells

Kaijun Luo 1,1, a , Matthew W Turnbull 1,b
PMCID: PMC3281302  PMID: 21521140

Abstract

Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca2+ and Mg2+ in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins.

Keywords : gap junction, hemichannel, hemocyte, innexin

Introduction

Gap junctions provide direct transfer of small molecules between adjacent cells in multicellular animals. Although functionally conserved across broad phylogeny, gap junctions are encoded by two multigene families: connexins are restricted to chordates, while the pannexin gene family encodes junctional proteins in both vertebrates and invertebrates. Pannexins, which were recently identified in mammalian genomes (Panchin et al. 2000), are referred to in insects and other invertebrates as innexins (invertebrate connexins). Connexin-based gap junctions consist of a pair of connexons or hemichannels, each provided by a single apposing cell of the pair, and each of which is comprised by six monomeric connexins (Yeager and Harris. 2007; Unger et al. 1999). Mammalian genomes encode approximately 20 connexins and 3 pannexins genes, which exhibit gene- and tissue-specific expression patterns (Willecke et al. 2002; Panchin et al. 2000). Insect genomes encode multiple innexin loci as well. For example, the Drosophila melanogaster genome encodes eight innexin loci (Stebbings et al. 2002), which display distinct expression patterns (Phelan et al. 1998a, 1998b; Stebbings et al. 2002; Lehmann et al. 2006) and a lack of functional redundancy (Curtin et al. 2002).

Gap junctions aid in the coordination of multicellular activities through the selective transfer of cytoplasmic metabolites, including nucleotides, ions such as Ca2+, lipid derivatives, and small peptides (Harris 2001). Connexin gap junction intercellular communication is involved in many physiological processes including left-right axis patterning (Levin 2007; Levin and Mercola. 1998), coordination of cell replication and death (Doble and Kardami 1995; Kalvelyte et al. 2003), antigen presentation in immunocytes (Neijssen et al. 2005), and neuronal adhesion and migration (Elias et al. 2007). Considering the likely evolutionary homology of pannexins and innexins (Baranova et al. 2004; Yen and Saier. 2007), they may share physiological roles or at least provide insight into one another. The roles of pannexin-mediated junctional communication are less clear than those that are connexin-mediated, but may include neural synchronization (Barbe et al. 2006). Gap junction-mediated communication is important in a wide range of insect physiological processes including epithelial morphogenesis (Bauer et al. 2004) and organogenesis (Bauer et al. 2002; Bauer et al. 2001), oocyte survival and maturation (Gilboa et al. 2003; Waksmonski and Woodruff. 2002), and activity of electrical synapses (Phelan et al. 1996) and Malpighian tubules (Weng et al. 2008).

The occurrence and function of nonjunctional (unapposed) hemichannels are less clear than those of gap junctions. Nonjunctional connexin hemichannels may coordinate cellular behaviors via paracrine signaling (e.g., by ATP and glutamine release) (Stout et al. 2002; Zhao et al. 2005), as well as affecting aggregation and adhesion (Cotrina et al. 2008). The activity levels of nonjunctional connexin hemichannels can change dynamically based on cellular conditions and the environment including pH and presence and concentration of certain ions (Contreras et al. 2001; De Vuyst et al. 2006, 2007; Retamal et al. 2007; Zhang et al. 2006). In contrast to connexins, the primary role of pannexins may be to generate nonjunctional hemichannels (Dahl and Locovei. 2006), and innexins have been demonstrated to form functional nonjunctional channels in leech neuronal tissue (Bao et al. 2007). It therefore seems likely that nonjunctional hemichannels may provide paracrine-mediated coordination of cellular activities in invertebrates, analogous to the hemichannels of mammals.

Gap junctions and/or innexin expression occurs in a wide array of insect tissues. Given the breadth of expression, the activity of nonjunctional hemichannels in leech tissue, and the functional similarities between innexins, pannexins, and connexins, it is highly likely that nonjunctional hemichannels occur in insects and may represent an as yet unreported communication modality. To address this possibility, we examined two lepidopteran cell lines and hemocytes for nonjunctional hemichannel activity using dye uptake assays. We observed activity of nonjunctional hemichannels in all three cell types and have noted the efficacy of several common pharmacological inhibitors on these hemichannels, which will be useful for future functional analyses. This work will facilitate future studies to examine the roles of insect hemichannels in insect biology, and comparisons to the chordate pannexins.

Materials and Methods

Cell culture and hemocyte isolation

High Five (BTI-TN-5B1-4), from Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) and Sf9 cells (from Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) were obtained from Invitrogen Corp. (http://www.invitrogen.com) and maintained as adherent cells in TnMFH media (Mediatech, www.cellgro.com) supplemented with 5% FBS. Cells were grown to confluency, harvested and washed 3 × 5 min in either TnMFH serum free media (SFM), or in Hanks' buffered salt solution (HBSS: 0.4 g/L KCl, 0.06 g/L KH2PO4, 8 g/L NaCl, 0.0477 g/L Na2HPO4, 0.35 g/L NaHCO3, 1 g/L glucose, pH 7.1) less Mg2+ and Ca2+ (divalent cation free, DCF), or HBSS plus 0.8 mM Mg2+ and 1.3 mM Ca2+ (divalent cation containing, DCC). Cell number and viability counts were performed prior to all assays using standard trypan blue staining and hemocytometer protocols. Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) larvae were obtained from Dr. Linda Gahan (Clemson University) and maintained as previously detailed (Gould et al. 1995), or were obtained commercially as second instar larvae (Bio-Serv, www.bio-serv.com). Fourth instar larvae were bled from an incision of abdominal proleg into serum-free TnMFH containing 200 µg/ml reduced glutathione to block melanization. Hemolymph was pooled from twenty larvae, washed with TnMFH SFM, and cell number and viability determined.

Dye uptake assays at 4°C

Dye uptake assays were performed to visualize the activity of nonjunctional hemichannels, using a variety of fluorescent dyes with different physical and chemical characteristics (Table 1). Stock solutions of ethidium bromide (EB, 0.5 mg/ml; Sigma, www.sigmaaldrich.com), 4′-6-diamidino-2-phenylindole (DAPI, 30 mM; Sigma), propidium iodide (PI, 1 mg/ml; Sigma), and lucifer yellow (LY, dilithium salt, 4 mg/ml; Sigma) were prepared in sterile ddH2O. Cells prepared as above were seeded in 96-well plates at 104 cells/well and incubated at 4 °C for 2 h, then 300 nM of DAPI, 5 µg/ml of EB, 50 µg/ml of PI, or 200 µg/ml LY fluorescence dye was added, dependent on assay design. After incubation at 4 °C for 5 min (cells were incubated in LY for 4 hrs, as discussed below), cells were washed 5 times with cold PBS, and fixed for 15 min in 3.7% formaldehyde. Cells were analyzed using fluorescence microscopy (below).

Table 1.

Characteristics and uptake of six fluorescent dyes in three different insect cell types at 4°C and room temperature.

graphic file with name t01_01.jpg

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Dye uptake assays at room temperature (23°C)

Rhodamine dextran (RX), Sigma) was prepared as a 10 mg/ml stock solution in sterile ddH2O. Calcein Blue AM (Invitrogen) was prepared as a 5 mM stock solution in DMSO. High Five, Sf9, and hemocytes prepared as above were transferred to a 96well tissue culture plate at 104 cells per well in appropriate media (TnMFH SFM, HBSS DCF, HBSS DCC). For analysis by flow cytometry, LY and RD were added to 60 µg/ml and 100 µg/ml, respectively. The plate was incubated with gentle rocking at room temperature for 1 h, and then cells were examined by flow cytometry (below). For fluorescence microscopy, LY was added to 200 µg/ml and the plate incubated with gentle rocking at room temperature for 4 hrs, prior to washing cells with the appropriate media; a longer incubation period and higher concentration were required with LY to conclusively distinguish dye uptake by microscopy. Suspected pharmacological modifiers of hemichannel activity were also tested. In those assays, cells were incubated for 30 min at room temperature in media containing carbenoxelone (CBX, 10 mM stock in sterile ddH2O; Sigma), flufenamic acid (FFA, 10 mM stock in DMSO; Sigma), mefloquine (MFO, 10 mM stock in 10% DMSO; Sigma), or lipopolysaccharide (E. coli LPS, 1 mg/ml stock in sterile ddH2O; Sigma), washed with TnMFH or HBSS, fresh media including dye was added, and cells processed as follows.

Flow cytometry

Flow cytometry was performed using a Guava EasyCyte Plus System, Millipore (www.millipore.com). Unlabeled High Five or Sf9 cells were used to gate fluorescence for either cell line, respectively. LY is gap junction permeable, while neither LY nor 10 kDa RD are membrane permeant, and RD is not gap junction permeant. Therefore, LY/RD- cells were considered unloaded, LY+/RD- cells were considered to have active hemichannels, LY-/RD+ considered to be endocytically active, and LY+/RD+ considered to be nonviable. The proportion of cells with active hemichannels was calculated as the ratio of (LY+/RD-) cells / total viable cells. Five thousand events were captured per replicated treatment, and each assay was repeated with at least three independent replicates.

Fluorescence microscopy

For microscopic analysis of dye uptake, cells were stained with calcein blue AM (2.5 µM final concentration) for 5 min at room temperature to verify viability following chemical presentation and dye loading. Samples were examined using a Nikon TE2000 inverted epifluorescence microscope equipped with DAPI, FITC, and TRITC filter sets and a Nikon DS2 monochrome camera. Samples stained with a single fluorophore (calcein blue AM, LY, or RD) were initially visualized to ensure lack of spectral overlap for paired fluorophores. A minimum of 125 cells and three non-overlapping fields of view were imaged per well, and three independent replications were performed for each treatment. Images were analyzed using Nikon Elements 2.0 (www.nikon.com). The proportion of cells with active hemichannels was calculated as the ratio of cells with both LY and calcein blue AM to cells with calcein blue AM only.

Statistical analyses

Statistical comparisons of experimental means to control means were performed using independent samples 2-tailed t-test with SPSS 16.0 for Windows (SPSS Inc.).

Results

Lepidopteran cells take up hemichannel permeant dyes

The uptake of dyes which are non-membrane permeable by cells can be indicative of unapposed hemichannel activity, although hemichannels may also be selectively permeable to dyes. Therefore the ability of lucifer yellow (LY), DAPI, propidium iodide (PI), and ethidium bromide (EB), which have very limited membrane permeability, were assayed for uptake. The large, non-membrane permeable molecule rhodamine dextran 10 kDa (RD), was also used as a marker for phagocytic activity, as well as the membrane permeant marker calcein blue AM, as a viability marker. Based on similar studies with mammalian systems (e.g., Page et al., 1994), we predicted that LY uptake and cellular distribution might vary between homogeneous and heterogeneous cytoplasmic localization. On the other hand, hemichannel-mediated uptake of DAPI, PI, and EB was expected to result in homogeneous cytoplasmic distribution of fluorescence (Valiunas, 2002). LY uptake was observed in pilot investigations using fluorescence microscopy, although discrimination from background fluorescence was difficult. To optimize the assay further the effect of interval post-plating was examined on dye uptake in Sf9 and High Five cells. Cells exhibited little to no uptake of dyes (PI, EB, DAPI, and LY) greater than 12 h post-plating (data not shown), suggesting little to no activity of nonjunctional hemichannels. At fewer than 12 h after plating, both cell lines exhibited uptake of all four tested dyes. Therefore, all assays reported here used freshly passaged (<12 h) cells. Furthermore, as LY was difficult to differentiate from background after 1 h incubation, microscopic analysis of that dye (along with simultaneous incubation with RD to identify phagocytotically active cells) was performed after 4 h incubation, while other molecules (PI, EB, and DAPI) were incubated for 5 min (Table 1, Figure 1A).

Figure 1.

Figure 1.

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Lepidopteran cells exhibit dye uptake suggestive of nonfunctional hemichannel activity. (A) High Five, Sf9, and primary culture H. virescens hemocytes are capable of uptake of PI, EB, and DAPI at 4° C, suggesting that endocytosis is not responsible for dye uptake. Scale bar = 50 µm. (B) Lucifer yellow uptake at room temperature occurs in all three cell types, as visualized by fluorescence microscopy; Calcein Blue AM staining indicates viability of the cells. Scale bar = 20 µm. All assays were performed in TnMFH SFM. High quality figures are available online.

High Five and Sf9 cells were examined using flow cytometry for their ability to take up the membrane impermeant molecules LY and RD at room temperature, in TnMFH SFM. Sf9 cells exhibited a slightly higher percentage of LY-positive cells under control conditions than High Five cells (27±3.1% and 23±2.5%, respectively; n=3) (Table 2). Few cells were positive for RD in these assays (data not shown), suggesting that dye uptake was hemichannel- and not phagocytosis-mediated. Cells were then analyzed for dye uptake and morphological analysis at room temperature using fluorescence microscopy with LY and calcein blue AM; positive staining with the latter verified cell viability. A large percentage of cells were positive for both LY and calcein blue AM, suggestive of active nonjunctional hemichannels in many of the cells. LY staining frequently was punctate, reminiscent of LY uptake and cellular distribution in rat myocytes (Page et al., 1994). Approximately half of all hemocytes exhibited some level of LY uptake, as well (Table 2, Figure 1B). Although not quantified, it appears that while granulocytes exhibited ready dye uptake, plasmatocytes rarely did (Figure 1B). Fluorescence microscopy consistently resulted in a higher percentage of LY-positive cells than flow cytometry (Table 2), likely due to the increased concentration and incubation period used in the former.

Table 2.

Lucifer yellow uptake observed in High Five and Sf9 cell lines and isolated Heliothis virescens hemocytes, in TnMFH.

graphic file with name t02_01.jpg

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To rule out phagocytosis as a mode of uptake, phagocytosis was inhibited by performing a subset of assays at 4° C. LY, PI, EB, and DAPI all entered Sf9 and High Five cells at rates similar to those observed at room temperature (data not shown). This, combined with the rarity of RD-positive cells in flow cytometry assays, strongly suggests that endocytosis is not responsible for a significant portion of dye uptake in these assays.

Known pharmacological blockers of hemichannels inhibit dye uptake

Carbenoxelone, flufenamic acid, and mefloquine are potent gap junction inhibitors in mammalian systems, and carbenoxelone inhibits nonjunctional hemichannel activity in leech neuronal tissue (Bao et al. 2007). The ability of these agents to block dye uptake in insect cells was tested, both to further test the likelihood that hemichannels mediate dye uptake and to identify modifying reagents. Pre-incubation of cells with carbenoxelone significantly reduced the percentage of LY-positive cells in TnMFH for both lines (Figure 2A, B). Flufenamic acid led to a slight (Sf9) to significant (High Five) increase in dye uptake, while mefloquine did not alter hemichannel-mediated uptake of LY. Carbenoxelone consistently inhibited dye uptake in the two cultured lines, and was found to significantly reduce LY uptake in hemocytes at 100 µM (Figure 2C). Viability was indistinguishable between control and chemical treated cells at 100 µM for all three cell types (data not shown). Flufenamic acid and mefloquine were not tested in hemocytes, given their lack of inhibitory activity in cell culture and the sensitivity of hemocytes to carbenoxelone.

Figure 2.

Figure 2.

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Alteration of dye uptake activity following treatment of cells with the gap junction inhibitors carbenoxelone (CBX), flufenamic acid (FFA), and mefloquine (MFO). (A) High Five and (B) Sf9 cell lines were analyzed by flow cytometry, and exhibit differential response to the three blockers in TnMFH SFM. (C) Mean dye uptake in hemocytes following exposure to carbenoxelone. Data are representative of 3 replicates performed in TnMFH SFM, and variance is s.e.m. Means indicated by asterisk significantly differ from untreated control, p< 0.05. High quality figures are available online.

Media Ca2+ and Mg2+ affect dye uptake

Reduced extracellular Ca2+ concentration is correlated with increased hemichannel activity for several connexin-based hemichannels (Li et al. 1996; Verselis and Srinivas. 2008), and alteration of intracellular Ca2+ affects leech nonjunctional hemichannel activity (Bao et al. 2007). Hanks' buffered salt solution (HBSS), either with 1.3 mM Ca2+ and 0.8 mM Mg2+ (DCC) or without the two cations (DCF), was used to examine the effect of the cations on lepidopteran hemichannels. High Five and Sf9 cells were examined for LY uptake using flow cytometry in both DCF and DCC. Incubation of cells in DCC resulted in increased dye uptake for both lines relative to cells incubated in DCF (Figure 3A, B). H. virescens hemocytes also exhibited a significant increase in LY uptake in DCC relative to DCF, as demonstrated with fluorescence microscopy (Figure 3C, D), suggesting that cation presence may be a positive regulator of lepidopteran hemichannel activity.

Figure 3.

Figure 3.

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Ca2+ and Mg2+content of the media significantly affects LY uptake. (A) High Five, (B) Sf9, and (C) hemocytes were examined for LY uptake in 1.3 mM Ca2+ and 0.8 mM Mg2+ (DCC), and 0 mM Ca2+ and Mg2+ (DCF) HBSS. Bars represent the mean percent of LY positive cells, scored by (A– B) flow cytometry or (C) fluorescence microscopy. (D) Hemocytes incubated in DCC and DCF media. Scale = 20 µm. Data are presented as means with s.e.m., from three independent replicates. Asterisk indicates that the mean of DCC treated cells significantly differs from DCF treatment at p< 0.05. High quality figures are available online.

LPS reduces hemichannel activity

In mammals, inflammatory stimuli lead in some cases to altered gap junctional and hemichannel activity in immunocytes. As hemocytes are the primary cellular components of the lepidopteran immune response, the effect of E. coli lipopolysaccharide (LPS) on hemichannel activity was examined. The presence of LPS reduced LY uptake in a concentration-dependent fashion in all three cell types (Figure 4). Flow cytometry showed that High Five cells were relatively sensitive to the application, exhibiting significant decreases in hemichannel activity at lower concentrations (Figure 4A) than Sf9 (Figure 4B). Fluorescence microscopy showed that preincubation with LPS similarly reduced LY uptake by H. virescens hemocytes (Figure 4C). Although dye uptake was reduced in LPS-treated Sf9 and H. virescens hemocytes, neither significantly differed from untreated cells.

Figure 4.

Figure 4.

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LPS reduces dye uptake in a dose-dependent fashion. (A) High Five cells exhibit a significant decrease in dye uptake in the presence of LPS, while (B) Sf9 cells and (C) H. virescens hemocytes exhibit a non-significant reduction. Data were obtained by (A, B) flow cytometry and (C) fluorescence microscopy. Data are presented as means with s.e.m., from three independent replicates. Means indicated by asterisk significantly differ from untreated control, p< 0.05. High quality figures are available online.

Discussion

Although gap junction intercellular communication is important in a wide variety of insect physiological processes (e.g., electrical synapse activity, organogenesis, yolk transfer, gamete viability) (Krishnan et al. 1993; Phelan et al. 1996; Bauer et al. 2001; Tazuke et al. 2002; Waksmonski and Woodruff. 2002), the activity and roles of nonjunctional hemichannels in insects have not been reported. However, leeches, which lack connexins, exhibit nonjunctional hemichannel activity as demonstrated by dye uptake and electrophysiological assays (Bao et al. 2007), suggesting that insects may have active nonjunctional hemichannels as well. Therefore three lepidopteran cell types were tested to examine the presence and activity of insect hemichannels. High Five cells, which are derived from T. ni, exhibit several characteristics of lepidopteran immunocytes (granulocytes), and are an in vitro model for hemocyte behavior (Beck and Strand. 2003). Sf9 cells are derived from pupal ovaries of S. frugiperda, and exhibit many endothelial characteristic; they have previously been used for heterologous expression of connexins (Beahm et al. 2006; Oshima et al. 2003; Stauffer 1995). The hemocytes isolated from H. virescens represent a mixed population of plasmatocytes and granulocytes, the primary immunocytes, as well as several minor populations (Lavine and Strand. 2002; Strand 2008; Davies et al. 1987). All three cell types have previously been demonstrated to form gap junctions and/or express innexins (Bukauskas et al. 1997; Epstein and Gilula. 1982; Turnbull et al. 2005). These study cells therefore include a range of cell origin and phenotype, and should provide a basic understanding of the distribution of nonjunctional hemichannels among several physiological systems. The findings, that lepidopteran cells exhibit nonjunctional hemichannel activity and that commonly used pharmacological and biological agents may affect their activity, are important in considering communication modalities in relevant physiological systems.

Partial inactivation of dye uptake by carbenoxelone was observed, but not for flufenamic acid and mefloquine, with effects varying by cell type and chemical concentration (Figure 2). Such variation may be due to the relative sensitivities of the constituent proteins (i.e., the sensitivity may be dependent on the particular constituent innexins), as is seen in the rabbit retina (Pan et al., 2007). Variability also was observed in response to inhibitors between pannexins and connexins: pannexins are relatively insensitive to flufenamic acid, while it is a potent blocker of the connexins (Pelegrin and Surprenant, 2006).

The Ca2+ and Mg2+ content of the media was found to have a strong effect on LY uptake. Increasing intracellular Ca2+ increased leech innexin nonjunctional hemichannel activity (Bao et al. 2007), while the presence of extracellular divalent cations generally is correlated with inhibition of connexin hemichannel activity (Verselis and Srinivas. 2008; Pfahnl and Dahl. 1999). Our results suggest a complex sensitivity that is potentially dependent on the innexin composition of the hemichannel, as has been postulated to be a basis for variation between mammalian gap junction sensitivities (Verselis and Srinivas. 2008). Additionally, our assays were generally performed in TnMFH SFM, which has Ca2+ and Mg2+ concentrations (9 mM and 2.2 mM, respectively) in the physiologically relevant range for these cell types. Although this Ca2+ level is higher than that reported for some lepidopterans, the Mg2+ concentration is much lower than that reported (Bindokas and Adams. 1988). The relatively high Mg2+ and Ca2+ levels in lepidopteran hemolymph, and the response of hemichannels to ion level, suggests there may be unique regulators of hemichannel activity relative to ion levels for lepidopterans. This possibility remains to be tested. But, regardless, the tested ion concentrations (in TnMFH and HBSS), coupled with our assay method (i.e., an endpoint analysis of dye uptake, rather than rate or concentration analysis), may explain the relatively high basal hemichannel activity found.

Until recently it was thought that nonjunctional hemichannels typically must reside in closed state, to avoid loss of cell homeostasis (Bennett et al. 2003). We utilized longer duration studies and found that a significant portion of the population of all three cells have at least intermittently open hemichannels. Although incapable of discriminating the duration of open state or the percent of open hemichannels, our methods verified that pharmacological and endotoxin modifiers are capable of sustained inhibition. Rate comparison studies are currently underway to refine this view of opened versus closed states.

Previous studies in mammalian (as reviewed in (Neijssen et al. 2007; Saez et al. 2000)) and insect systems (Churchill et al. 1993; Grimstone et al. 1967; Turnbull et al. 2005) have identified gap junctions or their encoding proteins in immunocyte populations. Interestingly, we found that all three investigated cell types exhibited a reduction in hemichannel activity following LPS application, a common model for immune stimulation. In mammalian leukocytes, LPS application may lead to connexin expression levels being down-regulated (De Maio et al. 2000; Temme et al. 2000; Fernandez-Cobo et al. 1999), up-regulated (Temme et al. 2000), unaffected (Oviedo-Orta et al. 2000), or post-translationally modified (De Maio et al. 2000; Gingalewski et al. 1996). Multiple innexins are expressed by the cells examined here (Turnbull et al., unpublished data), and it seems probable, by analogy to connexins and pannexins, that different innexins are differentially affected by inflammatory state. Leech hemichannels release ATP to activate and recruit microglia, immune cells of the central nervous system, to the site of injury (Samuels et al., 2010). Many signal transduction pathways involved in immune responses are broadly conserved phylogenetically (Gupta, 1991a, 1991b; Zakarian et al. 2003; Schmidt et al. 2008; Strand 2008), and we propose that hemichannels may play a similar role in regulating insect hemocyte behavior. Testing of this hypothesis is currently underway.

The identity of molecules transferred through nonjunctional hemichannels is rarely known (Schalper et al. 2008), although paracrine signaling by hemichannel-mediated transfer of small signaling compounds, such as ions, cyclic nucleotides and ATP, can be important (Stout et al. 2002; Zhao et al. 2005; Locovei et al. 2006). In preliminary examinations of High Five cells, no significant alteration in extracellular ATP levels was observed following LPS application (Luo, preliminary data), despite reduction in dye uptake. However, a more rigorous examination of possible signaling molecules including ATP and other likely candidates (Ca2+, Mg2+, cyclic nucleotides, lipid derivates, etc.) must be pursued. Additionally, large molecules may be capable of traversing insect gap junctions (Cieniewicz and Woodruff. 2008), suggesting that previously unconsidered molecules may also utilize hemichannels.

In conclusion, we have demonstrated for the first time the activity and subsequent modification of nonjunctional hemichannels in insect cells. Given the widespread use of insect cells for recombinant protein expression, and the functional analogy between both hemichannels and gap junctions comprised by innexins, pannexins, and connexins, these data support the use of insect cells in future studies of gap junction biology. Identification of the specific components transferred by hemichannels, though difficult, should be a key goal. In addition, characterizing the role of hemichannels in specific physiological systems of insects, particularly during ontogenic processes and immune responses, should also inform fundamental research into modes of cellular communication in those systems.

Acknowledgements

Critical comments were provided during this work and on the manuscript by Drs. Brad Hersh, Roland Hilgarth, and Tamara McNealy. Thanks also to Dr. Linda Gahan for providing caterpillars. This work was supported by a USDA-NRI award (200603787) to MWT and the National Natural Science Foundation of China (NSFC) (31060251) to Kaijun Luo.

Abbreviations

CBX,

carbenoxelone;

DAPI,

4′-6-Diamidino-2-phenylindole;

DCC,

divalent cation containing;

DCF,

divalent cation free;

EB,

ethidium bromide;

FFA,

flufenamic acid;

HBSS,

Hank's buffered salt solution;

LPS,

lipopolysaccharide;

LY,

lucifer yellow;

MFO,

mefloquine;

PI,

propidium iodide;

RD,

rhodamine dextran;

SFM,

serum free media

References

  1. Bao L, Samuels S, Locovei S, Macagno ER, Muller KJ, Dahl G. Innexins form two types of channels. FEBS Letters. 2007;581:5703–5708. doi: 10.1016/j.febslet.2007.11.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y. The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics. 2004;83:706–716. doi: 10.1016/j.ygeno.2003.09.025. [DOI] [PubMed] [Google Scholar]
  3. Barbe MT, Monyer H, Bruzzone R. Cell-cell communication beyond connexins: the pannexin channels. Physiology (Bethesda) 2006;21:103–114. doi: 10.1152/physiol.00048.2005. [DOI] [PubMed] [Google Scholar]
  4. Bauer R, Lehmann C, Hoch M. Gastrointestinal development in the Drosophila embryo requires the activity of innexin gap junction channel proteins. Cellular Adhesion and Communication. 2001;8:307–310. doi: 10.3109/15419060109080743. [DOI] [PubMed] [Google Scholar]
  5. Bauer R, Lehmann C, Fuss B, Eckardt F, Hoch M. The Drosophila gap junction channel gene innexin 2 controls foregut development in response to Wingless signaling. Journal of Cell Science. 2002;115:1859–1867. doi: 10.1242/jcs.115.9.1859. [DOI] [PubMed] [Google Scholar]
  6. Bauer R, Lehmann C, Martini J, Eckardt F, Hoch M. Gap junction channel protein Innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo. Molecular Biology of the Cell. 2004;15:2992–3004. doi: 10.1091/mbc.E04-01-0056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, Toloue MM, Chandrasekhar A, Nicholson BJ, Sosinsky GE. Mutation of a conserved threonine in the third transmembrane helix of a- and b-connexins creates a dominant-negative closed gap junction channel. Journal of Biological Chemistry. 2006;281:7994–8009. doi: 10.1074/jbc.M506533200. [DOI] [PubMed] [Google Scholar]
  8. Beck M, Strand MR. RNA interference silences Microplitis demolitor bracovirus genes and implicates glcl.8 in disruption of adhesion in infected host cells. Virology. 2003;314:521–535. doi: 10.1016/s0042-6822(03)00463-x. [DOI] [PubMed] [Google Scholar]
  9. Bennett MV, Contreras JE, Bukauskas FF, Saez JC. New roles for astrocytes: gap junction hemichannels have something to communicate. Trends in Neuroscience. 2003;26:610–617. doi: 10.1016/j.tins.2003.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bindokas VP, Adams ME. Hemolymph composition of the tobacco budworm, Heliothis virescens F. (Lepidoptera: noctuidae). Comparative Biochemistry and Physiology Part A : Physiology. 1998;90:151–155. [Google Scholar]
  11. Bukauskas FF, Vogel R, Weingart R. Biophysical properties of heterotypic gap junctions newly formed between two types of insect cells. Journal of Physiology. 1997;499:701–713. doi: 10.1113/jphysiol.1997.sp021962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Churchill D, Coodin S, Shivers RR, Caveney S. Rapid de novo formation of gap junctions between insect hemocytes in vitro: a freeze-fracture, dye-transfer and patch-clamp study. Journal of Cell Science. 1993;104:763–772. [Google Scholar]
  13. Cieniewicz AM, Woodruff RI. Importance of molecular configuration in gap junctional permeability. Journal of Insect Physiology. 2008;54:1293–1300. doi: 10.1016/j.jinsphys.2008.06.012. [DOI] [PubMed] [Google Scholar]
  14. Contreras JE, Sanchez HA, Eugenin EA, Speidel D, Theis M, Willecke K, Bukauskas FF, Bennett MV, Saez JC. Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proceedings of the National Academy of Science, U.S.A. 2001;26:26. doi: 10.1073/pnas.012589799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cotrina ML, Lin JH, Nedergaard M. Adhesive properties of connexin hemichannels. Glia. 2008;56:1791–1798. doi: 10.1002/glia.20728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Curtin K, Zhang Z, Wyman R. Gap junction proteins are not interchangeable in development of neural function in the Drosophila visual system. Journal of Cell Science. 2002;115:3379–3388. doi: 10.1242/jcs.115.17.3379. [DOI] [PubMed] [Google Scholar]
  17. Dahl G, Locovei S. Pannexin: to gap or not to gap, is that a question? IUBMB Life. 2006;58:409–419. doi: 10.1080/15216540600794526. [DOI] [PubMed] [Google Scholar]
  18. Davies DH, Strand MR, Vinson SB. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology. 1987;33:143–153. [Google Scholar]
  19. De Maio A, Gingalewski C, Theodorakis NG, Clemens MG. Interruption of hepatic gap junctional communication in the rat during inflammation induced by bacterial lipopolysaccharide. Shock. 2000;14:53–59. doi: 10.1097/00024382-200014010-00010. [DOI] [PubMed] [Google Scholar]
  20. De Vuyst E, Decrock E, Cabooter L, Dubyak GR, Naus CC, Evans WH, Leybaert L. Intracellular calcium changes trigger connexin 32 hemichannel opening. EMBO Journal. 2006;25:34–44. doi: 10.1038/sj.emboj.7600908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. De Vuyst E, Decrock E, De Bock M, Yamasaki H, Naus CC, Evans WH, Leybaert L. Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor. Molecular Biology of the Cell. 2007;18:34–46. doi: 10.1091/mbc.E06-03-0182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Doble BW, Kardami E. Basic fibroblast growth factor stimulates connexin-43 expression and intercellular communication of cardiac fibroblasts. Molecular and Cellular Biochemistry. 1995;143:81–87. doi: 10.1007/BF00925930. [DOI] [PubMed] [Google Scholar]
  23. Elias LAB, Wang DD, Kriegstein AR. Gap junction adhesion is necessary for radial migration in the neocortex. Nature. 2007;448:901–908. doi: 10.1038/nature06063. [DOI] [PubMed] [Google Scholar]
  24. Epstein ML, Gilula NB. A study of communication specificity between cells in culture. Journal of Cell Biology. 1982;75:769–787. doi: 10.1083/jcb.75.3.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fernandez-Cobo M, Gingalewski C, Drujan D, De Maio A. Downregulation of connexin 43 gene expression in rat heart during inflammation. The role of tumour necrosis factor. Cytokine. 1999;11:216–224. doi: 10.1006/cyto.1998.0422. [DOI] [PubMed] [Google Scholar]
  26. Gilboa L, Forbes A, Tazuke SI, Fuller MT, Lehmann R. Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state. Development. 2003;130:6625–6634. doi: 10.1242/dev.00853. [DOI] [PubMed] [Google Scholar]
  27. Gingalewski C, Wang K, Clemens MG, De Maio A. Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation. Journal of Cellular Physiology. 1996;166:461–467. doi: 10.1002/(SICI)1097-4652(199602)166:2<461::AID-JCP25>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  28. Gould F, Anderson A, Reynolds A, Bumgarner L, Moar W. Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins. Journal of Economic Entomology. 1995;88:1545–1559. [Google Scholar]
  29. Grimstone AV, Rotheram S, Salt G. An electron-microscope study of capsule formation by insect blood cells. Journal of Cell Science. 1967;2:281–292. doi: 10.1242/jcs.2.2.281. [DOI] [PubMed] [Google Scholar]
  30. Gupta AP. Gap cell junctions, cell adhesion molecules, and molecular basis of encapsulation. In: Gupta AP, editor. Immmunology of Insects and Other Arthropods. CRC Press; 1991a. pp. 133–167. [Google Scholar]
  31. Gupta AP. Insect immunocytes and other hemocytes: roles in cellular and humoral immunity. In: Gupta AP, editor. Immunology of Insects and Other Arthropods. CRC Press; 1991b. pp. 21–118. [Google Scholar]
  32. Harris AL. Emerging issues of connexin channels: biophysics fills the gap. Quarterly Review of Biophysics. 2001;34:325–472. doi: 10.1017/s0033583501003705. [DOI] [PubMed] [Google Scholar]
  33. Kalvelyte A, Imbrasaite A, Bukauskiene A, Verselis VK, Bukauskas FF. Connexins and apoptotic transformation. Biochemical Pharmacology. 2003;66:1661–1672. doi: 10.1016/s0006-2952(03)00540-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krishnan SN, Frei E, Swain GP, Wyman RJ. Passover: a gene required for synaptic connectivity in the giant fiber system of Drosophila. Cell. 1993;73:967–977. doi: 10.1016/0092-8674(93)90274-t. [DOI] [PubMed] [Google Scholar]
  35. Lavine M, Strand M. Insect hemocytes and their role in immunity. Insect Biochemistry and Molecular Biology. 2002;32:1295–1309. doi: 10.1016/s0965-1748(02)00092-9. [DOI] [PubMed] [Google Scholar]
  36. Lehmann C, Lechner H, Loer B, Knieps M, Herrmann S, Famulok M, Bauer R, Hoch M. Heteromerization of innexin gap junction proteins regulates epithelial tissue organization in Drosophila. Molecular Biology of the Cell. 2006;17:1676–1685. doi: 10.1091/mbc.E05-11-1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Levin M. Gap junctional communication in morphogenesis. Progress in Biophysics and Molecular Biology. 2007;94:186–206. doi: 10.1016/j.pbiomolbio.2007.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Levin M, Mercola M. Gap junctions are involved in the early generation of left-right asymmetry. Developmental Biology. 1998;203:90–105. doi: 10.1006/dbio.1998.9024. [DOI] [PubMed] [Google Scholar]
  39. Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG. Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. Journal of Cell Biology. 1996;134:1019–1030. doi: 10.1083/jcb.134.4.1019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Locovei S, Bao L, Dahl G. Pannexin 1 in erythrocytes: Function without a gap. Proceedings of the National Academy of Science, USA. 2006;103:7655–7659. doi: 10.1073/pnas.0601037103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Neijssen J, Herberts C, Drijfhout JW, Reits E, Janssen L, Neefjes J. Cross-presentation by intercellular peptide transfer through gap junctions. Nature. 2005;434:83–88. doi: 10.1038/nature03290. [DOI] [PubMed] [Google Scholar]
  42. Neijssen J, Pang B, Neefjes J. Gap junction-mediated intercellular communication in the immune system. Progress in Biophysics and Molecular Biology. 2007;94:207–218. doi: 10.1016/j.pbiomolbio.2007.03.008. [DOI] [PubMed] [Google Scholar]
  43. Oshima A, Doi T, Mitsuoka K, Maeda S, Fujiyoshi Y. Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function. Journal of Biological Chemistry. 2003;278:1807–1816. doi: 10.1074/jbc.M207713200. [DOI] [PubMed] [Google Scholar]
  44. Oviedo-Orta E, Hoy T, Evans WH. Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations. Immunology. 2000;99:578–590. doi: 10.1046/j.1365-2567.2000.00991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Page E, Goings GG, Upshaw-Earley J, Hanck DA. Endocytosis and uptake of Lucifer yellow by cultured atrial myocytes and isolated intact atria from adult rats. Circulation Research. 1994;75:335–346. doi: 10.1161/01.res.75.2.335. [DOI] [PubMed] [Google Scholar]
  46. Pan F, Mills SL, Massey SC. Screening of gap junction antagonists on dye coupling in the rabbit retina. Visual Neuroscience. 2007;24:609–618. doi: 10.1017/S0952523807070472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Panchin Y, Kelmanson I, Matz M, Lukyanov K, Usman N, Lukyanov S. A ubiquitous family of putative gap junction molecules. Current Biology. 2000;10:R473–474. doi: 10.1016/s0960-9822(00)00576-5. [DOI] [PubMed] [Google Scholar]
  48. Pelegrin P, Surprenant A. Pannexin-1 mediates large pore formation and interleukinlβ release by the ATP-gated P2X7 receptor. EMBO Journal. 2006;25:5071–5082. doi: 10.1038/sj.emboj.7601378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pfahnl A, Dahl G. Gating of cx46 gap junction hemichannels by calcium and voltage. Pflugers Archives. 1999;437:345–353. doi: 10.1007/s004240050788. [DOI] [PubMed] [Google Scholar]
  50. Phelan P, Bacon JP, Davies JA, Stebbings LA, Todman MG, Avery L, Baines RA, Barnes TM, Ford C, Hekimi S, Lee R, Shaw JE, Starich TA, Curtin KD, Sun YA, Wyman RJ. Innexins: a family of invertebrate gap-junction proteins. Trends in Genetics. 1998a;14:348–349. doi: 10.1016/s0168-9525(98)01547-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Phelan P, Nakagawa M, Wilkin MB, Moffat KG, O'Kane CJ, Davies JA, Bacon JP. Mutations in shaking-B prevent electrical synapse formation in the Drosophila giant fiber system. Journal of Neuroscience. 1996;16:1101–1113. doi: 10.1523/JNEUROSCI.16-03-01101.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Phelan P, Stebbings LA, Baines RA, Bacon JP, Davies JA, Ford C. Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes. Nature. 1998b;391:181–184. doi: 10.1038/34426. [DOI] [PubMed] [Google Scholar]
  53. Retamal MA, Schalper KA, Shoji KF, Bennett MV, Saez JC. Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential. Proceedings of the National Academy of Science, U.S.A. 2007;104:8322–8327. doi: 10.1073/pnas.0702456104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Saez JC, Branes MC, Corvalan LA, Eugenin EA, Gonzalez H, Martinez AD, Palisson F. Gap junctions in cells of the immune system: structure, regulation and possible functional roles. Brazilian Journal of Medical and Biological Research. 2000;33:447–455. doi: 10.1590/s0100-879x2000000400011. [DOI] [PubMed] [Google Scholar]
  55. Samuels SE, Lipitz JB, Dahl G, Muller KJ. Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury. Journal of General Physiology. 2010;136:425–442. doi: 10.1085/jgp.201010476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schalper KA, Palacios-Prado N, Orellana JA, Saez JC. Currently used methods for identification and characterization of hemichannels. Cell Communication and Adhesion. 2008;15:207–218. doi: 10.1080/15419060802014198. [DOI] [PubMed] [Google Scholar]
  57. Schmidt O, Theopold UH, Beckage NE. Insect and vertebrate immunity: Key similarities versus differences. In: Beckage NE, editor. Insect Immunology. Academic Press; 2008. pp. 1–24. [Google Scholar]
  58. Stauffer KA. The gap junction proteins beta 1-connexin (connexin-32) and beta 2-connexin (connexin-26) can form heteromeric hemichannels. Journal of Biological Chemistry. 1995;270:6768–6772. [PubMed] [Google Scholar]
  59. Stebbings LA, Todman MG, Phillips R, Greer CE, Tarn J, Phelan P, Jacobs K, Bacon JP, Davies JA. Gap junctions in Drosophila: Developmental expression of the entire innexin gene family. Mechanisms of Development. 2002;113:197–205. doi: 10.1016/s0925-4773(02)00025-4. [DOI] [PubMed] [Google Scholar]
  60. Stout CE, Costantin JL, Naus CC, Charles AC. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. Journal of Biological Chemistry. 2002;14:14. doi: 10.1074/jbc.M109902200. [DOI] [PubMed] [Google Scholar]
  61. Strand MR. Insect hemocytes and their role in immunity. In: Beckage NE, editor. Insect Immunology. Academic Press; 2008. pp. 25–48. [Google Scholar]
  62. Tazuke SI, Schulz C, Gilboa L, Fogarty M, Mahowald AP, Guichet A, Ephrussi A, Wood CG, Lehmann R, Fuller MT. A germline-specific gap junction protein required for survival of differentiating early germ cells. Development. 2002;129:2529–2539. doi: 10.1242/dev.129.10.2529. [DOI] [PubMed] [Google Scholar]
  63. Temme A, Ott T, Haberberger T, Traub O, Willecke K. Acute-phase response and circadian expression of connexin26 are not altered in connexin32- deficient mouse liver. Cell and Tissue Research. 2000;300:111–117. doi: 10.1007/s004410000177. [DOI] [PubMed] [Google Scholar]
  64. Turnbull MW, Volkoff A-N, Webb BA, Phelan P. Functional gap-junction genes are encoded by insect viruses. Current Biology. 2005;15:R491–492. doi: 10.1016/j.cub.2005.06.052. [DOI] [PubMed] [Google Scholar]
  65. Unger VM, Kumar NM, Gilula NB, Yeager M. Three-dimensional structure of a recombinant gap junction membrane channel. Science. 1999;283:1176–1180. doi: 10.1126/science.283.5405.1176. [DOI] [PubMed] [Google Scholar]
  66. Valiunas V. Biophysial properties of connexin-45 gap junction hemichannels studied in vertebrate cells. Journal of General Physiology. 2002;119:147–164. doi: 10.1085/jgp.119.2.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Verselis VK, Srinivas M. Divalent cations regulate connexin hemichannels by modulating intrinsic voltage-dependent gating. Journal of General Physiology. 2008;132:315–327. doi: 10.1085/jgp.200810029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Waksmonski SL, Woodruff RI. For uptake of yolk precursors, epithelial celloocyte gap junctional communication is required by insects representing six different orders. Journal of Insect Physiology. 2002;48:667–675. doi: 10.1016/s0022-1910(02)00095-1. [DOI] [PubMed] [Google Scholar]
  69. Weng XH, Piermarini PM, Yamahiro A, Yu MJ, Aneshansley DJ, Beyenbach KW. Gap junctions in Malpighian tubules of Aedes aegypti. Journal of Experimental Biology. 2008;211:409–422. doi: 10.1242/jeb.011213. [DOI] [PubMed] [Google Scholar]
  70. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G. Structural and functional diversity of connexin genes in the mouse and human genome. Biological Chemistry. 2002;383:725–737. doi: 10.1515/BC.2002.076. [DOI] [PubMed] [Google Scholar]
  71. Yeager M, Harris AL. Gap junction channel structure in the early 21st century: Facts and fantasies. Current Opinion in Cell Biology. 2007;19:521–528. doi: 10.1016/j.ceb.2007.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Yen MR, Saier MH., Jr. Gap junctional proteins of animals: the innexin/pannexin superfamily. Progress in Biophysics and Molecular Biology. 2007;94:5–14. doi: 10.1016/j.pbiomolbio.2007.03.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zakarian RJ, Dunphy GB, Rau ME, Albert PJ. Kinases, intracellular calcium, and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect, Galleria mellonella. Archives of Insect Biochemistry and Physiology. 2003;53:158–171. doi: 10.1002/arch.10097. [DOI] [PubMed] [Google Scholar]
  74. Zhang X, Zou T, Liu Y, Qi Y. The gating effect of calmodulin and calcium on the connexin50 hemichannel. Biological Chemistry. 2006;387:595–601. doi: 10.1515/BC.2006.076. [DOI] [PubMed] [Google Scholar]
  75. Zhao HB, Yu N, Fleming CR. Gap junctional hemichannel-mediated ATP release and hearing controls in the inner ear. Proceedings of the National Academy of Science, U.S.A. 2005;102:18724–18729. doi: 10.1073/pnas.0506481102. [DOI] [PMC free article] [PubMed] [Google Scholar]

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