Efficient transfection of NG108-15 cells with GABA(B) receptor subunits was achieved using polyethylenimine. Baclofen modulated high voltage-activated Ca(2+) current in differentiated cells transfected with GABA(B1) and GABA(B2) receptor subunits or with the GABA(B2) subunit alone, but not with the GABA(B1) subunit alone. Characteristics of the current modulation were very similar for cells transfected with GABA(B1/2) and GABA(B2) subunits. Using antisense oligonucleotides against GABA(B1) subunits and also western immunoblotting, we are able to show that NG108-15 cells contain endogenous GABA(B1) subunits. Therefore, functional receptors can be formed by the combination of native GABA(B1) subunits with transfected GABA(B2) subunits, in agreement with the proposed heteromeric structure of GABA(B) receptors. Finally, we used selective channel blockers to identify the subtypes of Ca(2+) channels that are modulated by GABA(B) receptors. In fact, in differentiated NG108-15 cells, the recombinant GABA(B) receptors couple only to N-type Ca(2+) channels.

Activation of gamma-aminobutyric acid(B) (GABA(B)) receptors in dorsal root ganglion (DRG) neurones leads to inhibition of calcium (Ca2+) channels. The role of the GABA(B1) receptor subunit was assessed by its depletion achieved by microinjection of DRG neurones with an antisense (A/S) oligodeoxynucleotide (ODN). Control neurones were injected with a scrambled version of the A/S ODN (missense) or were not injected. Patch clamp recordings of Ca2+ channel current were made two to four days after injection. GABA(B1) A/S substantially reduced the current inhibition induced by baclofen, a GABAB agonist. Therefore, most, if not all, native GABAB receptors which couple to Ca2+ channels contain GABA(B1). Moreover, if native receptors are heterodimers of GABA(B1) and GABA(B2), then GABA(B2), in isolation, is unable to sustain coupling to Ca2+ channels. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Two GABA(B) receptor subunits have been cloned: GABA(B1) and GABA(B2). In this study we investigate the coupling of recombinant GABA(B) receptors to calcium channels in differentiated NG108-15 cells, which exhibit many similarities to neurones but in which functional GABA(B) receptors are normally absent. Transfection of GABA(B1) and GABA(B2) subunit cDNAs enables baclofen-mediated inhibition of different calcium channel subtypes and a component of this modulation is voltage-dependent. When transfected individually, GABA(B2), but not GABA(B1), is able to enhance calcium current inhibition over background levels. Further, an antisense oligodeoxynucleotide to GABA(B1) reduces the average functional response in cells transfected with GABA(B2) alone. Assuming that the functional receptor is heteromeric, this suggests that GABA(B1), but not GABA(B2), is expressed endogenously in NG108-15 cells.

Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage‐dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF‐1 and FGF‐2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos. Long‐term exposure of the embryonic myocytes to FGF‐1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage‐dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF‐2 upregulated these currents, although, because of an increase in capacitance caused by FGF‐2, current density did not change with this factor. The regulation of IK(IR) by FGF‐1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF‐1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel‐blocking mechanism. In many cells, IK,S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage‐activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S. In summary, FGF‐1 and FGF‐2 exert opposite effects on voltage‐dependent K+ currents in embryonic myocytes and, furthermore, FGF‐1 achieves its effects on different K+ currents via separate second messenger pathways.

Single inward rectifier K+ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K+ channels (Kir(0.3), Kir(0.5) and Kir(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K+] o respectively. Extracellular Ba2+ blocked the normally high channel activity in a concentration-dependent manner (K A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K+ current, marked voltage dependence of Ba2+ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg2+-free solution at pH 9.1. This was probably also true for Kir(0.3), Kir(0.5) and Kir(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K+ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states.

Regulation of early embryonic expression of sodium current in cultured Xenopus laevis myocytes was investigated. In myocytes isolated before innervation and cultured for about 1 day, only 51% expressed sodium current at a mean density of 74 +/- 20 pA/pF (mean +/- SEM; n = 26), inadequately reflecting the functional development of skeletal muscle in vivo at this time. This cell-autonomous expression pattern could be modulated. First, co-culture of myocytes and dissociated neural tissue induced additional sodium current expression (82%; 172 +/- 31 pA/pF, n = 14) and, secondly, removal of calcium from the culture medium reduced sodium current density (13 +/- 6 pA/pF; n = 5). The former results suggest that a diffusible factor released from differentiating neurons is able to initiate the expression of sodium channels in myocytes.

The development of excitable cells is characterized by highly organized patterns of expression of ion channels. During the terminal differentiation Xenopus muscle somites, potassium currents are expressed first just after Stage 15 (early-mid neurula), following a long period during which no voltage-dependent currents can be detected in any cell in the dorsal embryo. We have investigated whether early expression of a foreign delayed rectifier potassium channel may affect this endogenous pattern of electrical development. We injected the purified cRNA of the mammalian brain Shaker-like potassium channel, Kv1.1, into fertilized Xenopus eggs. The resulting currents were analyzed in blastomeres during a 12-hr period prior to Stage 15 and in differentiating muscle cells after Stage 15. In injected embryos, a high fraction of blastomeres expressed a delayed rectifier-type current. The Kv1.1 current could be distinguished from the endogenous muscle delayed potassium current (IK,X) by its very different voltage dependence. Separation of currents based on this difference indicated that, in injected embryos, I K,X appeared much earlier in development than in control embryos. Furthermore, even in cells which expressed solely Kv1.1-type current, the sensitivity of the current to dendrotoxin declined dramatically during development, approaching that of I K,X . These data suggest an interaction between Kv1.1 and endogenous channel subunits, and/or modification of the Kv1.1 protein by the embryonic cells in ways not seen in Xenopus oocytes or mammalian cell lines.

Although the development of several of the voltage-dependent currents in embryonic amphibian myocytes has been described, the overall muscle electrical development, particularly the relative times of expression of different voltage-dependent currents, has not been addressed in a single study under one set of conditions. We have found that, in mesoderm isolated and cultured from neurula stage embryos, myocytes are identifiable before they express voltagegated currents. These ionic currents are absent from all Xenopus mesodermal cells during the late gastrula/early neurula stages of embryonic development. At about the time of first somite segregation an inward rectifier K+ current is expressed in some myocytes, followed within 2 hr by a delayed rectifier K+ current. The density of both currents increases fourfold over the next 24 hr in culture. A Na+ current is not expressed in large numbers of myocytes until late in this culture period, at about the time that a slow Ca2+ current appears. Under our culture conditions the myocytes have a very low chloride conductance. A fast inactivating component to the outward K+ current is expressed in all myocytes by 24 hr in culture. In some experiments we dissociated embryos at later times and made recordings when all previously isolated myocytes expressed currents. In the late dissociations, most myocytes did not express currents, but developed them after a short period in culture. Because we have evidence that in vivo development is more closely approximated by the early dissociations, these results suggest that dissociation causes some degree of dedifferentiation.

Annals of the New York Academy of SciencesVolume 635, Issue 1 p. 318-327 Millisecond Studies of Single Membrane Fusion Eventsa W. ALMERS, W. ALMERS Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorL. J. BRECKENRIDGE, L. J. BRECKENRIDGE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.Search for more papers by this authorA. IWATA, A. IWATA Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorA. K. LEE, A. K. LEE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorA. E. SPRUCE, A. E. SPRUCE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 Department of Zoology, University of Washington, Seattle, WA 98195.Search for more papers by this authorF. W. TSE, F. W. TSE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this author W. ALMERS, W. ALMERS Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorL. J. BRECKENRIDGE, L. J. BRECKENRIDGE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.Search for more papers by this authorA. IWATA, A. IWATA Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorA. K. LEE, A. K. LEE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this authorA. E. SPRUCE, A. E. SPRUCE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195 Department of Zoology, University of Washington, Seattle, WA 98195.Search for more papers by this authorF. W. TSE, F. W. TSE Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195Search for more papers by this author First published: October 1991 https://doi.org/10.1111/j.1749-6632.1991.tb36502.xCitations: 21 a This work was supported by NIH Grants AR-17803 and GM-39520. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume635, Issue1Calcium Entry and Action at the Presynaptic Nerve TerminalOctober 1991Pages 318-327 RelatedInformation

During exocytosis, secretory vesicles of mast cells generate a current transient that marks the opening of the fusion pore, the first aqueous connection that forms between the vesicle lumen and the cell exterior. By recording and analyzing such current transients, we have tracked the conductance of the fusion pore over the first millisecond of its existence. The first opening of the pore occurs rapidly, generally within 100 microseconds at 23 degrees C. The electric conductance of the pore is a few hundred picosiemens at first, but gradually increases over the subsequent milliseconds. Evidently the pore opens abruptly and then dilates. The initial conductance of the pore suggests a diameter comparable to that of a large ion channel. From an analysis of "capacitance flicker" we infer that a pore can increase its diameter severalfold and still close again completely. This suggests that several early events in membrane fusion are reversible.