Journal of Cell Science 112, 2391-2396 (1999) 2391 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0478 Innexin-3 forms connexin-like intercellular channels Yosef Landesman1, Thomas W. White2, Todd A. Starich3, Jocelyn E. Shaw3, Daniel A. Goodenough2 and David L. Paul1,* Departments of 1Neurobiology and 2Cell Biology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115, USA 3Department of Genetics and Cell Biology, University of Minnesota, 1445 Gortner Ave., St Paul, MN 55108, USA *Author for correspondence (e-mail: [email protected]) Accepted 12 May; published on WWW 24 June 1999 SUMMARY Innexins comprise a large family of genes that are believed induces electrical coupling between the oocyte pairs. In to encode invertebrate gap junction channel-forming addition, analysis of INX-3 voltage and pH gating reveals proteins. However, only two Drosophila innexins have been a striking degree of conservation in the functional directly tested for the ability to form intercellular channels properties of connexin and innnexin channels. These data and only one of those was active. Here we tested the ability strongly support the idea that innexin genes encode of Caenorhabditis elegans family members INX-3 and EAT- intercellular channels. 5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, Key words: Innexin, inx-3, eat-5, Intercellular channel, Gap junction INTRODUCTION expression of the of the Drosophila shakingB(lethal) in paired Xenopus oocytes induced intercellular channel formation Regulated cell-cell communication is essential for normal although expression of an alternate splice form, development and proper tissue homeostasis. A widely utilized shakingB(neural) did not. form of communication is enabled by intercellular channels Since only one Drosophila innexin has been directly contained in gap junctions. In vertebrates, these channels are demonstrated to form intercellular channels, we have tested the composed of connexins, and they permit the diffusion of ability of two C. elegans family members, INX-3 and EAT-5 low molecular mass compounds between adjacent cells to form intercellular channels in oocyte pairs. EAT-5 is (Goodenough et al., 1996). Analysis of connexin gene expressed in the pharynx and synchronized contraction of knockouts and naturally occurring connexin mutations indicate pharyngeal muscles is lost in the eat-5 mutants (Starich et al., that gap junctional communication has diverse functions in the 1996). In addition, dye coupling between pharyngeal myocytes development and physiological activity of several organ pm5 and pm4, readily detectable in wild-type animals, is systems (White and Paul, 1999). Gap junctional absent in the mutant. Loss of both synchrony and dye coupling communication has also been well documented in many are consistent with disruption of junctional communication, invertebrate organisms (de Laat et al., 1980; Warner and which could coordinate excitation by electrically connecting Lawrence, 1982; Weir and Lo, 1985; Blennerhassett and the muscle cells. Ectopic expression of EAT-5 rescues both the Caveney, 1984; Fraser et al., 1987) and is altered in several uncoordinated pharyngeal muscle contraction and the reduced Drosophila and C. elegans mutants (Sun and Wyman, 1996; dye-coupling. The inx-3 gene (Starich et al., 1996, Phelan et al., 1996; Starich et al., 1996). Analysis of these wEST01007) is expressed in multiple locations including the mutants has revealed a family of genes unrelated to connexins isthmus and terminal bulb regions of the pharynx, partially that could serve the same function, a possibility supported by overlapping the pharyngeal expression of EAT-5. Thus far, Inx- the fact that no invertebrate connexin genes have been 3 mutants have not been isolated and its biological significance identified. Originally termed OPUS for founding members remains to be determined. Here we report that INX-3, but not ogre, passover, unc-7 and shakingB (Barnes, 1994; Starich et EAT-5, induces intercellular communication between paired al., 1996), the family has been renamed innexin (innexin = Xenopus oocytes. No functional interaction between the two invertebrate connexin) (Phelan et al., 1998a). There are no innexins can be detected in our expression system, suggesting primary sequence relationships between connexins and that they operate independently. innexins although both gene families encode four transmembrane domain proteins. Currently two Drosophila and more than twenty four C. elegans innexin genes have been MATERIALS AND METHODS identified (Barnes and Hekimi, 1997). Support for the hypothesis that innexins can form intercellular channels was Xenopus oocytes, RNA preparation and microinjection recently provided by Phelan et al. (1998b) who showed that Oocytes were isolated and microinjected first with a specific antisense 2392 Y. Landesman and others oligonucleotide to deplete endogenouse Cx38 and the following day junctional conductance to recover. Although no attempt was made to these oocytes were injected with synthetic RNA as previously determine the precise levels of pHi under these conditions, described (Paul et al., 1995). For in vitro transcription of RNA, the measurements with both pH-sensitive microelectrodes and proton- respective coding regions of inx-3 and eat-5 were subcloned into sensitive dyes indicate that, in oocytes, this procedure typically pCS2+ (Turner and Weintraub, 1994). Constructs were linearized with induces a rapid intracellular acidification in excess of 1 pH unit NotI and used as template for SP6 RNA polymerase (mMESSAGE (Morley et al., 1997; Wang and Peracchia, 1997). Values of junctional mMACHINE kit, Ambion). conductance were determined in response to alternating ±10 mV pulses applied at 15 second intervals for the entire length of the In vivo synthesis and labeling protocol, and normalized to the conductance values recorded prior to For metabolic labelling, oocytes were microinjected with 60 ng the start of perfusion with 100% CO2. synthetic RNA and were incubated in groups of 5 oocytes for 6-12 hours in 500 µl of modified Barth’s medium with 50 µCi [35S]methionine (NEG-009T, New England Nuclear, Boston, MA). Total proteins were prepared by homogenization of 5 oocytes in 50 RESULTS µl of RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton, 0.1% SDS, 1% sodium deoxycholate and 5 mM EDTA) for Expression of INX-3 and EAT-5 in Xenopus oocytes separation by SDS-PAGE. Membrane fractions were prepared as To assess oocyte synthesis of EAT-5 and INX-3, proteins were previously described (Paul et al., 1995). labelled by incubating RNA injected oocytes in medium Antibodies containing [35S]methionine. Whole cell extracts were The anti-INX-3 antiserum was prepared by injection of a C-terminal subjected to SDS-PAGE and analysed by autoradiography. fusion protein into rabbits (T. A. Starich and J. E. Shaw, unpublished INX-3 and EAT-5 migrated in SDS-PAGE as 44 and 46 kDa results) and was diluted 1:500 to detect ectopic expression of INX-3 proteins, respectively (Fig. 1A, specific bands indicated by on blots containing membrane fractions from Xenopus oocytes. white arrows). These values were close to the values predicted by cDNA analysis (48.9 and 50.3 kDa, respectively). Similarly, Electrophysiology positive controls consisting of vertebrate connexins (Cx38 and To characterize gap junctional channels, oocytes were stripped of the Cx43) produced products with expected SDS-PAGE migration. vitelline envelope in hypertonic medium (Methfessel et al., 1986) and Steady state levels of INX-3 and EAT-5 were similar to those paired with the vegetal poles facing each other. Electrophysiological analysis by dual electrode voltage clamp (Spray et al., 1981) was of connexin gene products in RNA injected oocytes. As a performed 48 hours after injection of RNAs to record the coupling further confirmation that INX-3 was synthesized correctly, values reported in Table 1. The data included in Fig. 2 were acquired western blotting with anti-INX-3 antiserum was performed. A at lower conductances (~1 µS) 24 hours after RNA injection. Current band with the expected SDS-PAGE mobility was detected in and voltage electrodes were pulled to a resistance of 1-2 MΩ with a membrane fractions of oocytes expressing INX-3 but not in vertical puller (David Kopf Instruments, Tujunga, CA) and filled with water injected controls or oocytes expressing Cx43 (Fig. 1B). a solution containing 3 M KCl, 10 mM EGTA and 10 mM Hepes, pH 7.4. Voltage clamping was performed using two GeneClamp 500 INX-3 induces the formation of gap junctions in amplifiers (Axon Instruments, Foster City, CA) controlled by an Xenopus oocyte pairs archaic IBM-PC compatible computer through a Digidata 1200 Since INX-3 and EAT-5 accumulate in Xenopus oocytes after interface (Axon Instruments). C-Lab II software (Indec System, Sunnyvale, CA) was used to program stimulus and data collection RNA injection, we tested their ability to form intercellular routines. The two cells of a pair were clamped at −40 mV, close to channels in oocyte pairs. After RNA injection, oocytes were their initial resting potential, which ranged between −40 and −50 mV. stripped of their vitelline envelopes and manipulated together For conductance measurements, 10 mV depolarizing steps were to form pairs. After an appropriate period (see Materials and applied alternately to each of the cells. Under these conditions, the Methods), each cell was impaled with two microelectrodes