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FEBS Letters 581 (2007) 5703–5708

Innexins form two types of channels

Li Baoa,c,1, Stuart Samuelsa,b,1, Silviu Locoveia,1, Eduardo R. Macagnoc, Kenneth J. Mullera,b, Gerhard Dahla,b,* a Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 016430, Miami, FL 33136, United States b Neuroscience Program, University of Miami, Miami, FL 33136, United States c Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, United States

Received 19 September 2007; revised 7 November 2007; accepted 9 November 2007

Available online 21 November 2007

Edited by Maurice Montal

release [7,8]. But calcium waves can occur in a free Abstract Injury to the central nervous system triggers glial cal- cium waves in both vertebrates and invertebrates. In vertebrates environment. The waves are not restricted to vertebrates but the pannexin1 ATP-release channel appears to provide for cal- are found in a broad spectrum of phyla that include inverte- cium wave initiation and propagation. The , which form brates [9–11]. The invertebrate organisms do not express con- invertebrate gap junctions and have sequence similarity with the nexins, but the unrelated family of pannexins, are candidates to form non-junctional membrane proteins [12,13]. channels. Two leech innexins previously demonstrated in glia Recent evidence indicates that pannexins [14], a second fam- were expressed in frog oocytes. In addition to making gap junc- ily of vertebrate gap junction proteins that exhibit sequence tions, innexins also formed non-junctional membrane channels homology to innexins, are a plausible alternative to with properties similar to those of pannexons. In addition, car- as the ATP release channel [6]. We therefore tested whether benoxolone reversibly blocked the loss of carboxyfluorescein innexins, like vertebrate pannexins, are capable of forming dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins channels in the non-junctional membrane. Different leech (Hir- we assayed. udo sp.) innexins, expressed by glial cells or by neurons, were Ó 2007 Federation of European Biochemical Societies. Pub- expressed in Xenopus oocytes and analyzed by whole cell volt- lished by Elsevier B.V. All rights reserved. age clamp and patch-clamp.

Keywords: Pannexin; Innexin; Calcium wave; ATP; Glia; Evolution 2. Materials and methods

2.1. Oocytes Oocytes were prepared as described previously [15]. In brief, Xeno- pus laevis oocytes were isolated by incubating small pieces of ovary in 2 mg mlÀ1 collagenase in calcium free oocyte Ringer’s solution 1. Introduction (OR2) and stirring at 1 turn/second for 3 h at room temperature. After being thoroughly washed with regular OR2, oocytes devoid of follicle Intercellular calcium waves are widespread and, in addition cells and having uniform pigmentation were selected and stored in to signaling injury to the nervous system, serve important OR2 at 18 °C. physiological functions, including synchronization of ciliary 2.2. In vitro transcription of mRNAs beats, modulation of synaptic transmission, ossification, and Hirudo innexins 2 and 3 (Hminx2 and Hminx3) had been cloned into control of vascular perfusion [1–4]. Calcium waves involve the expression vector pCR-BluntII-TOPO. mRNA was transcribed by gap junction channels for propagation from cell to cell [1]. SP6 (Hminx2) or T7 (Hminx3) RNA polymerase from 10 lg of XbaI- However, they also involve the release of ATP from the cyto- (Hminx2) or SpeI- (Hminx3) linearized plasmid using the mMessage plasm to the extracellular space [5]. Binding of ATP to P2 mMachine kit (Ambion). mRNA was quantified by absorbance (260 nm), and the proportion of full-length transcripts was checked receptors results in further ATP release and propagation of by agarose gel electrophoresis. Twenty nanoliters of mRNA (50 ng/ the wave. Several mechanisms for ATP release have been pro- ml) was injected into oocytes. posed, including vesicular release and channel-mediated re- lease through a variety of membrane channels [6]. Although 2.3. Solutions a large body of literature suggests that a non-junctional mem- OR2 solution in mM: 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 brane channel with properties of gap junction ‘‘hemichannels’’ Na2HPO4, 5.0 HEPES, antibiotics (Penicillin, 10000 U/ml; Streptomy- cin, 10 mg/ml, pH 7.5). Patch pipette KGlu solution: 140 mM potas- represents the ATP release channel, the identity of the channel sium gluconate, 10 mM KCl, 5.0 mM TES, pH 7.5). is questionable. Various vertebrate gap junction proteins, in particular connexins 43 and 32, have been implicated in ATP 2.4. Voltage clamp Whole cell voltage clamp recording was performed with two intra- cellular microelectrodes as described [15]. *Corresponding author. Address: Department of Physiology and Biophysics, University of Miami, School of Medicine, P.O. Box 016430, Miami, FL 33136, United States. Fax: +1 305 243 5931. 2.5. Patch-clamp technique E-mail address: [email protected] (G. Dahl). Single innexon channels were studied by the patch-clamp technique [16] using a WPC-100 amplifier (E.S.F. Electronics, Goettingen, Ger- 1These authors contributed equally to this paper. many). Currents were filtered at 5 kHz, digitized using a VR-10B

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.030 5704 L. Bao et al. / FEBS Letters 581 (2007) 5703–5708

digital data recorder, and stored on video tape. The recordings were -40 mV transferred to a Power Macintosh (Apple) computer using an ITC- a -20 mV 0 mV 18 Computer Interface (Instrutech Corporation) and analyzed. Acqui- 50 nA sition and analysis were done with the Acquire and TAC programs 2 min (both from Bruxton Corporation, Seattle, WA). Details of patch-clamp techniques used, including application of Inx2 negative pressure, can be found elsewhere [17,18]. 2.5.1. Extracellular ATP measurements from oocytes. ATP assay b gm (μS) solutions (Luciferin/Luciferase, Sigma, St. Louis) were mixed with supernatants collected from hm-inx2 injected and uninjected cells trea- 20 ted with oocyte Ringer’s solution (OR2) or potassium gluconate (KGlu) in the presence of 0–100 lM carbenoxolone (CBX). Oocytes 15 were 4 days post-injection. Innexin expression and cell viability were confirmed electrophysiologically. Cells were pretreated for 10 min in 10 experimental solutions and then isolated for 10 min in 150 lL of the same experimental solutions. 100 lL supernatant was obtained for 5 each condition. Each condition was done in quintuplicate. Lumines- cence readings were obtained with a Victor 1420 multilabel counter 0 (PerkinElmer, Waltham, MA) on a 96 well culture plate. ANOVA with -40 -30 -20 -10 0 10 20 Post-Hoc Fisher test (Statistica, StatSoft Inc., Tulsa, OK) was used for mV comparison and statistical analysis of luminescence. c d 2.5.2. In situ dye release. 6-Carboxyfluorescein was injected into a -10 mV -10 mV Inx3 single glia cell of the leech connective and dye release was followed un- Inx2 50 nA 50 nA der a fluorescence microscope. Details of the technique are given in 2 min 2 min Supplement 1 and Supplementary Fig. 1.

intracellular acidification intracellular acidification e 3. Results

100nA 3.1. Innexins form innexons 1min 35 min Both Hminx2 and Hminx3 formed patent ‘‘hemichannels’’ carbenoxolone (innexons) in the non-junctional plasma membrane norm. luminescence (Fig. 1a,b). Like pannexin 1, but in contrast to ‘‘hemichannels’’ f 50 formed by some connexins, no change of extracellular calcium *** was required to observe innexon channel activity. The channels 40 were closed at the resting (À40 mV) and opened when the membrane was depolarized to À20 mV or ** 30 higher (Fig. 1a,b). Cytoplasmic acidification, induced by per- * fusing the oocytes with CO2-saturated Ringer’s solution, closed the channels (Fig. 1c,d). This effect slowly reversed upon 20 * washout (Supplementary Fig. 2). Moreover, carbenoxolone closed the channels in a dose-dependent way (Fig. 1e and Sup- 10 plementary Fig. 3). In Fig. 1b, as shown previously [19], sham injected oocytes did not exhibit a current that was activated in uninjected innexin 2 expressing oocytes this voltage range and that was sensitive to cytoplasmic acidi- OR2 KGlu OR2 KGlu fication. CBX (μM) To test whether the formation of innexons might be a gen- 1 5 10 20 50 100 eral property of innexins, two other innexins, Hminx1 and Fig. 1. Macroscopic membrane currents. (a): The membrane potential Hminx6, which are found in leech neurons, were also expressed of an Hminx2-expressing oocyte was held at the potentials indicated in oocytes. Both formed non-junctional membrane channels and 5 mV hyperpolarizing voltage pulses to test membrane resistance with properties similar to those of the channels formed by were applied (top trace) at a rate of 6/min. The corresponding currents are shown in the bottom trace. (b) Voltage dependence of membrane Hminx2 and Hminx3 (Supplementary Fig. 4). conductance (gm) of oocytes expressing Hminx2 (m), Hminx3 (.)or To test for ATP permeability of innexons, the efflux of ATP uninjected oocytes ( ). Means ± S.D. (n = 4, 5, or 5, respectively) are from oocytes expressing Hminx2 was measured by a lumino- plotted. The membrane potential was held at the respective voltages metric luciferase assay. As reported previously, uninjected con- for 5 min and conductance was determined with 5 mV depolarizing trol oocytes released some ATP when depolarized by a high pulses.(c,d) Application of CO2 to the bath solution reduced membrane currents of oocytes expressing Hminx2 (c) or Hminx3 (d). potassium solution (KGlu) (Fig. 1f), [18,20] which probably (e) Addition of 100 lM carbenoxolone increased membrane resistance represents the Brefeldin sensitive vesicular release [20]. Hminx2 and blocked the current in an oocyte expressing Hminx2. Note expressing oocytes had the same basal release as control oo- the slow time course of recovery. (f) ATP release mediated by cytes. However, when depolarized with high potassium solu- Hminx2is inhibited by carbenoxolone. Release of ATP to the extra- cellular medium by oocytes was measured by luminometry using a tion a significant increase in ATP release was observed luciferase assay. Depolarization of the cells with high potassium (Fig. 1f). Carbenoxolone inhibited this ATP release in a solution (KGlu) resulted in a small ATP release from uninjected dose-dependent way (Fig. 1f). oocytes and a significantly larger release from Hminx2injected oocytes. Consistent with the macroscopic membrane currents, expres- Carbenoxolone attenuated this larger release in a dose-dependent *** ** sion of Hminxs1, 2, 3 and 6 in single Xenopus oocytes resulted fashion. Means ± S.E.M. are given (n = 5). P < 0001, P < 0.001, P* < 0.05. in the appearance of a novel type of , with a L. Bao et al. / FEBS Letters 581 (2007) 5703–5708 5705 large unitary conductance of about 500 pS (Hminxs2, 3 and 6) 3.2. Innexon channels are mechanosensitive and about 250 pS (Hminx1) in symmetrical 150 mM KCl It has been reported that pannexon channels are sensitive to (Fig. 2a and Supplementary Fig. 4). The channels were closed mechanical stress [18]. To test whether innexons are also mech- at negative potentials and open at positive potentials. The volt- anosensitive, we used cell-attached and excised membrane age gate was slow, and consequently the channel remained patches from oocytes expressing Hminx2. After identification open for tens of seconds after switching from positive to of the innexons, based on single channel conductance, voltage negative potential. The channel activity was attributable to dependence and gating properties (archetypal subconductance innexins because such large conductance channels were not levels), the channels were subjected to mechanical stress by observed in control oocytes and because of the distinctive suction applied to the patch pipette [21]. Fig. 2b shows a typ- features of the channel, which were quite similar to pannexin ical response of Hminx2 channels to stretch. When the patch, 1 channels expressed in oocytes [18]. Like pannexin 1 channels, containing at least three channels, was held at À50 mV, all leech innexin channels tested here, when active, exhibited mechanical stress caused activity to increase. The channel multiple subconductance states at which they mainly dwelled, activity initially was in the form of subconductance states be- while sojourns to the full open and closed states were infre- fore full openings were discerned. For quantitative analysis, quent (Fig. 2a and Supplementary Fig. 4). For a more detailed open probability was determined before and during stretch characterization of the channels we concentrate hereafter on (Fig. 2b). During prolonged stretch, channel activity often sub- those formed by Hminx2. sided spontaneously (not shown).

Fig. 2. Single channel currents of leech Hminx2. (a) The membrane potential was held at positive potential to activate the channel before shifting to À50 mV. Like human pannexin 1 channels, the channel has a large unit conductance (500 pS in 150 mM KCl) and exhibits many subconductance levels (compare to Fig. 1 in [17]). Histogram shows distribution of current amplitudes, with intermediate peaks at subconductance levels. (b) Patch- clamp recording from a membrane patch of an oocyte exogenously expressing Hminx2. After activation of the channels at +20 mV, the membrane potential was held at À50 mV. At this potential, channel activity continued for a period of time and then subsided, as was typical for this holding potential. Negative pressure (40 mbar) was applied by suction to the patch pipette during the time indicated by the line. That the patch contained at least three channels can be seen by the steps during negative pressure application. 5706 L. Bao et al. / FEBS Letters 581 (2007) 5703–5708

Oocytes are known to express an endogenous stretch acti- the dye was lost from the connective every 5 min. This dye loss vated channel [22–24]. However, the channel properties includ- was almost completely blocked in saline with 10 lM carbenox- ing unit conductance (50 pS), kinetics, and calcium block of olone. This effect was reversed by rinsing the carbenoxolone this endogenous channel [17,24] are distinct from those of the from the tissue, as shown for a representative example in large conductance channel seen only in innexin mRNA in- Fig. 3a. Fig. 3b shows the average of 6 experiments. Carbenox- jected oocytes (Fig. 2). olone’s attenuation of dye release demonstrated that non-junc- tional channels were involved in the passage of dye across the membrane. 3.3. Innexon channels are activated by cytoplasmic calcium Pannexin1 channels can be activated by cytoplasmic cal- cium. We tested whether the functional similarity of pannexin and innexin channels also extend to this property. Oocytes endogenously express a calcium activated chloride channel a that could potentially interfere with the analysis. However, 200 the innexon channels can be discriminated easily from the endogenous calcium activated chloride channel based on their unitary conductances of 500 pS and 3 pS [25], respectively. After identification of innexon channels at potentials permis- sive for channel opening, the channels were held at À50 mV. At this potential, channels were closed or stayed in low sub- 100 conductance states. Application of calcium to the intracellular side of the channels contained in excised inside–out membrane patches resulted in channel activity that ceased upon washout of the calcium containing solution (Supplementary Fig. 5). CBX

Free calcium concentrations in the micromolar range were suf- fluorescence intens. (arbtrary units) ficient to elicit this effect. Current recordings from inside–out 0 50 100 150 200 patches containing multiple Hminx2 channels were used to Time (min) determine the Ca2+dose–response relationship (Supplementary 2+ À6 b Fig. 5b). When the free [Ca ] was 10 M or less, the channels 20 ** * opened briefly and resided in low subconductance states. When the free [Ca2+] was 10À5 M or higher, multiple channels opened, resulting in rapid increases in patch currents. Repeated applications of 10À5 M [Ca2+] to the intracellular side resulted in decreased current amplitudes and slower acti- vation kinetics (Supplementary Fig. 6). It is unclear whether this adaptation phenomenon was due to complete inactivation 10 of individual channels or whether the majority of channels were driven into lower subconductance states. dye loss (% per 5 min) 3.4. Innexons function in vivo Although measurements using frog oocytes show that Hminx1, 2, 3 and 6 form innexons, direct study of the leech nervous system is required to determine whether innexins nor- saline CBX wash 10 μM mally form innexons in vivo. One measure of the presence of pannexons and other ‘‘hemichannels’’ has been the uptake or c protostomes loss across the plasmalemma of small molecules of dye that can be specifically blocked by drugs such as carbenoxolone [26], which we showed above blocks Hminx2 innexon activity in frog oocytes (Fig. 1e,f). deuterostomes A single 5 mm-long, glial cell expressing both Hminx2 and Hminx3 ensheathes all the axons of each connective between innexon (innexins) ganglia along the nerve cord of the leech (Supplementary pannexon (pannexins) Fig. 1a) [27]. To determine whether Hminx2 and Hminx3 form connexon (connexins) non-junctional innexon channels in the living nervous system as they do in oocytes, dye release studies were done on live Fig. 3. Carbenoxolone reversibly inhibits 6-carboxyfluorescein dye loss from the connective glial cell. (a) Representative time course of dye connective glial cells. The dye 6-carboxyfluorescein (6-CF), loss from a 1 mm piece of 6-carboxyfluorescein (6-CF) injected which is gap junction permeable for leech glia, was directly in- connective glial cell (see Supplementary Fig. 1b). Carbenoxolone jected into the connective glial cell using small negative cur- (CBX, 10 lM) was added and washed out as indicated. (b) Quanti- rents (À2 nA). Injection was restricted to one of the two glial fication of average dye loss before, during, and after CBX treatment. * ** cells of the connective; the second served as control. Both cells ( P < .01, P < 0.001, n = 6). Error bars represent S.E.M. (c) A schematic diagram showing that innexins share the roles both of were imaged at 5 min intervals to determine the rate of dye loss pannexins in forming channels to the extracellular space and of (see Section 2). In leech saline solution, approximately 15% of connexins in forming gap junctions. L. Bao et al. / FEBS Letters 581 (2007) 5703–5708 5707

4. Discussion membrane channels. Our in vivo study supports the presence of innexons in the intact leech nervous system. Like pannexin These results indicate that Hminxs1, 2, 3 and 6, when 1 channels, the Hminx2 innexin channels described here have expressed in Xenopus oocytes, form functional innexons that qualities expected for the ATP release channel involved in ini- permit the exchange between cytoplasm and extracellular tiation and propagation of calcium waves. These include space of molecules in the size range of second messengers. This mechanosensitivity, activation by cytoplasmic calcium, and is in addition to the established role of innexins to form gap ATP permeability. We propose that with the acquisition of junction channels, which has been demonstrated for Hm-inx2 connexins by deuterostomes, the gap junction role was as- [28]. With only hints towards its existence [6,29], innexon chan- sumed by these proteins while the innexin orthologs in deuter- nel activity has not yet been described for innexins; previous ostomes, the pannexins, were retained for the non-junctional studies were mostly limited to addressing the fundamental channel function (Supplementary Fig. 5c). question of which protein participates in gap junctional func- tion in invertebrates [12,30]. Whether the capacity to form inn- Acknowledgements: We thank Dr. I. Dykes for preparing the Hminx2 exon channels is a general property of all innexins remains to and Hminx3 constructs. Supported in part by NIH training grants and a Lois Pope Fellowship (S.S.), an AHA graduate fellowship (S.L.), be determined, but it is of interest to note that all four leech NSF Grant 0446346 (E.R.M.) and NIH Grants NS34927 (K.J.M.), innexins thus far tested do. NS37025 (K.J.M.) and GM48610 (G.D.) and an AHA Grant (G.D.). The channels formed by the Hirudo sp. innexins share sev- eral properties with human and mouse pannexin 1 channels. The unitary conductances of the channels are similar, yet dis- tinct. In the full open state pannexin 1 channels have a single Appendix A. Supplementary data channel conductance of 550 pS in 150 mM potassium chloride solution [18]. Under identical conditions the single channel Supplementary data associated with this article can be conductance of Hminxs2, 3 and 6 channels is 500 pS and that found, in the online version, at doi:10.1016/j.febs- of Hminx1 is 250 pS. let.2007.11.030. Like pannexin 1 channels, the innexin channels exhibit mul- tiple subconductance levels and at voltages permissive for channel opening, excursions to the full closed as well to the full References open state are scarce. The single prominent subconductance [1] Sanderson, M.J., Charles, A.C. and Dirksen, E.R. 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