Journal of Cell Science 112, 2391-2396 (1999) 2391 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0478

Innexin-3 forms -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 channel-forming addition, analysis of INX-3 voltage and pH gating reveals . However, only two Drosophila 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 genes encode of 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 , these channels are demonstrated to form intercellular channels, we have tested the composed of , 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 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 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 (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 for current supplied by the clamp to the cell not stepped was equal in dual cell voltage clamping. This procedure permitted direct amplitude, but opposite in sign to the junctional current (Ij). Junctional quantitation of electrical conductance between the cells, a conductance was calculated by dividing the junctional current by the measure of intercellular communication (Spray et al., 1981). transjunctional potential. For analysis of voltage dependence, Possible contributions to background conductance by transjunctional potentials of opposite polarity were generated by endogenous Cx38 (Ebihara et al., 1989; Gimlich et al., 1990) hyperpolarizing or depolarizing one cell in 10 mV steps, while clamping the second cell at −40 mV. Following the imposition of each were eliminated in these experiments by pre-injection of the voltage step, steady state currents were measured 30 seconds after the oocytes with Cx38 anti-sense oligonucleotides (Barrio et al., onset of the voltage pulse, normalized to their value at ±10 mV, and 1991). As a result, background levels of conductance were plotted against the transjunctional potential. Data were fit to a below the level of detection (Table 1). Pairs of oocytes in which Boltzmann equation of the form: Gjss = (1−Gjmin)/{1+exp(A[V− both cells expressed INX-3 developed robust conductances Vo])}+Gjmin, where Gjss is the steady state conductance, Gjmin is similar in magnitude to control pairs expressing Cx43 (Table minimum conductance, A is the cooperativity constant and Vo is the 1). In contrast, pairs expressing EAT-5 did not develop voltage at which the decrease in Gjss is half maximal. Sensitivity of measurable conductances. Thus, either eat-5 is not a channel Gj to membrane potential (Vi-o) was tested as previously described forming protein or it is not active in Xenopus oocytes. Since (White et al., 1994). INX-3 and EAT-5 overlap in their patterns of expression, we The effect of intracellular proton concentration (pHi) on junctional conductance was tested on paired oocytes expressing INX-3. tested the possibility that EAT-5 could form active intercellular Following a brief equilibration period, the incubation dish (total channels in the presence of INX-3. However, pairs in which volume = 6 ml) was perfused for 5 minutes with MB solution saturated one cell expressed INX-3 and the other EAT-5 developed no with 100% carbon dioxide at a flow rate of 4 ml/minute, after which conductance above background (Table 1). In addition, the the perfusion medium was switched back to normal MB to allow coexpression of INX-3 and EAT-5 in the same oocytes did not Innexin-3 forms connexin-like intercellular channels 2393

Table 1. Electrical coupling in paired oocytes Junctional RNA injection conductance Number of kDa (cell 1/cell 2) (Gj) in µS pairs INX-3/INX-3 17±4 18 EAT-5/EAT-5 0 11 Cx43/Cx43 72±8.2 5 Water/water 0 13 INX-3/EAT-5 0 7

mV. At Vj greater than 20 mV, junctional current (Ij) decreased over time to a lower equilibrium value in >10 seconds. The sensitivity of INX-3 channels to transjunctional voltage and the relatively slow time course of their response were strikingly similar to connexins (Bennett and Verselis, 1992). These data were also consistent with the qualitative analysis of shakingB(lethal) voltage-dependence (Phelan et al., 1998b). A plot of the INX-3 conductance (Gj)-voltage relationship (Fig. 2B) further illustrated the similarity to connexin voltage gating. INX-3 channels displayed a substantial residual conductance (28%) that was not sensitive to transjunctional potential. The transjunctional potential at which conductance was half- kDa maximal (Vo) was 33 mV, a value in the middle of the range for connexin channels (reviewed by White et al., 1995b). In addition, the slope of the transition from maximum to minimum conductance was described by a cooperativity constant of A = 0.17, again a value well within the normal range of values for connexins. Finally, the sensitivity of INX-3 junctional conductance to changes in membrane potential (Vi-o), was measured by clamping paired oocytes at a series of different Fig. 1. Expression of INX-3 and EAT-5. (A) SDS-PAGE analysis of holding potentials (0-100 mV). Junctional currents at each [35S]methionine-labeled proteins from Xenopus oocytes injected with potential were measured by imposing brief 10 mV test pulses. water or with 60 ng of RNA encoding EAT-5, INX-3, Cx38 or Cx43. INX-3 channels were not sensitive to membrane potential (Fig. Each lane contains the equivalent of 1/2 cell from each sample. 2C), unlike the junctional currents measured in several insect Specific bands not in water-injected cells are indicated with white cell types (Verselis et al., 1991; Bukauskas and Peracchia, 1997; arrows. (B) Immunoblot containing insoluble material from Xenopus Gho, 1994; Churchill and Caveney, 1993). However, some oocytes injected with either 50 or 100 pg inx-3 RNA reacted with connexins are sensitive to this parameter while others are not anti-INX-3 antibodies. Control oocytes were either left uninjected or and it is possible that innexins will also exhibit a range of injected with 100 pg Cx43 RNA. INX-3 is marked with an arrow. A behaviors. A larger fraction of the twenty four C. elegans nonspecific band with a mobility slightly faster than INX-3 is visible innexin gene products (Barnes and Hekimi, 1997) will need to in all lanes. be tested to resolve this issue. A characteristic aspect of connexin chemical gating is alter significntly the levels of conductances compared to those sensitivity to pH (for review see Morley et al., 1997). The expressing INX-3 alone (data not shown). relationship between chemical gating of connexin and innexin channels was investigated by cytoplasmic acidification (Fig. 3). Electrical properties of INX-3 channels in paired Conductance between paired oocytes expressing INX-3 was Xenopus oocytes measured while the bathing solution was saturated with 100% Electrophysiological properties of intercellular channels joining CO2. Gj levels started to decline 2 minutes after exposure to insect cells have been extensively characterized and are similar CO2 and were reduced to 40% of their initial values after 5 to those of connexin channels in vertebrate cells (Verselis et al., minutes. The inhibition of conductance was reversible, and 1991; Bukauskas and Peracchia, 1997; Gho, 1994; Churchill following superfusion of the paired oocytes with normal buffer and Caveney, 1993). Thus, if innexins are the invertebrate for another 4 minutes, Gj returned to its initial value. Thus, counterparts of connexins, the gating properties of innexin innexin channel sensitivity to cytoplasmic acidification was channels should be similar to those of connexins. Therefore, we very similar to that observed for many connexins. characterized the voltage and pH gating of INX-3 channels. INX-3 channels displayed voltage-dependent closure when transjunctional potentials were applied (Fig. 2A). In this DISCUSSION experiment, both cells of the pair were initially held at −40 mV after which the membrane potential of one cell was changed in We have shown that INX-3, a C. elegans innexin, is competent 10 mV steps, resulting in transjunctional voltages (Vj) of 10-80 to form intercellular channels. The gating properties of its 2394 Y. Landesman and others A Fig. 2. Voltage gating properties of INX-3 channels. (A) The effect of transjunctional voltage (Vj) on the junctional currents (Ij) 100 nA developed by oocyte pairs expressing INX-3. Hyperpolarizing or depolarizing voltage steps were applied in 10 mV increments to one cell in a pair. At Vj greater than 20 mV, junctional currents decreased 10 s over the time of the voltage step (30 seconds) and the kinetics of closure increased with increasing Vj. (B) A plot of steady state junctional conductance (Gj) versus Vj. Steady state conductance was well fit by a Boltzmann equation with the following parameters: A = 0.17; Vo = 33 mV; Gjmin = 0.28. Data were derived from 3 cell pairs with a mean Gj = 2.97±0.45 µS. (C) INX-3 junctional conductance is independent of the membrane potential (Vi-o). Both cells in a pair were simultaneously clamped to a series of holding potentials (Vj = 0; Vi-o = −100, −80, −60, −40, −20, 0, +20, +40). Cells were equilibrated at a given potential for 60 seconds and then 10 mV test pulses were applied to one cell in the pair for 250 milliseconds to measure Gj. Gjs were normalized to their value at B Vi-o = −100 and plotted against Vi-o. Data are derived from 3 cell µ 1.2 pairs with a mean Gj=7.44±4.38 S.

1.0

100% CO2 0.8 1.2

0.6 1.0

normalized Gj 0.4 0.8

0.2 0.6

0.0 -80 -60 -40 -20 0 20 40 60 80 0.4 normalized Gj Vj [mV] 0.2 C

1.2 0.0 0 2 4 6 8 10 12 14 1.0 time [min] Fig. 3. INX-3 channels are gated by cytoplasmic pH. INX-3 channels 0.8 are sensitive to cytoplasmic acidification. Oocyte pairs were superfused with MB equilibrated with 100% CO2 (gray bar) and 0.6 conductances were normalized to their initial values. Coupling levels decreased 60% from their initial values within 5 minutes, and this reduction was fully reversible upon switching the perfusion medium normalized Gj 0.4 to normal buffer. Values plotted are the mean ± s.e.m. of three INX-3 injected pairs, which displayed a mean initial conductance of 0.2 2.8±1.2 µS.

0.0 -100 -80 -60 -40 -20 0 20 40 oocytes is not without precedent among gap junction proteins. Vi-o [mV] Neither Cx31.1, Cx33 nor the neural form of Drosophila shakingB produce active channels in oocyte pairs (Bruzzone et channels are indistinguishable from those of connexins with al., 1994; White et al., 1995a; Phelan et al., 1998b). One regard to the sensitivity and kinetics of voltage dependent possibility is that those proteins are constitutively inactive as closure, the characteristic residual conductance and the channels and perhaps have other functions. Another possibility response to cytoplasmic acidification. These data strongly is that they can form active channels, but need either another support the hypothesis that invertebrate intercellular channels connexin/innexin partner. As an example, Bruzzone et al. are encoded by innexin genes, and opens a powerful genetic (1994) reported a chimeric connexin that did not induce system to studies of intercellular communication in C. elegans. conductance when paired with itself but could contribute to The inability of EAT-5 to induce communication in Xenopus active intercellular channels when paired with a different Innexin-3 forms connexin-like intercellular channels 2395 connexin. Finally, it is possible that channel activity must be compartments by a cell type with reduced junctional permeability. Nature positively modulated by an accessory protein not present in the 309, 361-364. Xenopus oocytes, or that the activity of a negative modulator Bruzzone, R., White, T. W. and Paul, D. L. (1994). Expression of chimeric connexins reveals new properties of the formation and gating behavior of constituitively present in oocytes needs to be suppressed. As gap junction channels. J. Cell Sci. 107, 955-967. an example, phosphorylation of Cx43 by the src gene product Bukauskas, F. F. and Peracchia, C. (1997). Two distinct gating mechanisms completely abolishes its activity (Swenson et al., 1990). in gap junction channels Ð CO2-sensitive and voltage-sensitive. Biophys. J. Given the lack of sequence identity between connexins and 72, 2137-2142. Churchill, D. and Caveney, S. (1993). Double whole-cell patch-clamp innexins, it is surprising that the physiological properties of the characterization of gap junctional channels in isolated insect epidermal cell invertebrate and vertebrate intercellular channels are so similar. pairs. J. Membr. Biol. 135, 165-180. Both types of channels exhibit characteristic slow kinetics of de Laat, S. W., Tertoolen, L. G. J., Dorresteijn, A. W. C. and van den closure in response to transjunctional potential and a Biggelaar, J. A. M. (1980). Intercellular communication patterns are substantial residual current indicating a non-voltage dependent involved in cell determination in early molluscan development. Nature 287, 546-548. component. In addition, quantification of innexin voltage Doolittle, R. F. (1994). Convergent evolution: the need to be explicit. Trends. gating by fitting Boltzmann relations, revealed that for all Biochem. Sci. 19, 15-18. parameters, innexin values were in the middle of the range of Ebihara, L., Beyer, E. C., Swenson, K. I., Paul, D. L. and Goodenough, D. values derived for connexin channels (Fig. 2b). Why should A. (1989). Cloning and expression of a Xenopus embryonic . Science 243, 1194-1195. vertebrates and use what appear to be completely Fraser, S. E., Green, C. R., Bode, H. R. and Gilula, N. B. (1987). Selective different gene families to accomplish the same function? 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