Biophysics Voltage gating and permeation in a gap junction hemichannel ( connexins / intercellular communication / ion channels / permeability /

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each oftwo adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained fromXenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, Yopen, to a substate, Ysub, about one-third of the conductance of Yoen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, Yclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between yVcosed and Yopen appear slow (10-20 ms), often involving several transient substates distinct from Ysub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (-300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (-8.5 A diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46. Gap junctions channels are constructed as two hemichannels (connexons) in series, one provided by each of the coupled cells. These channels provide a direct connection for passage of ions between the coupled cells that is insulated from the extracellular space. Their permeability to relatively large molecules, up to -1 kDa in molecular mass, and presence in inexcitable cells suggest a role in transmission of chemical as well as electrical signals. The constituent hemichannels are hexamers of protein subunits of the connexin gene family, which has at least 12 members in mammals (1). One issue in gap junction channel physiology is the functional significance of this diversity, which requires characterization of the channels in terms of gating and permeability. However, characterization of gating and permeability of these channels is hampered by their inaccessibility to direct patch recording. Although the double whole cell patch technique has been used extensively in single channel studies, junctional conductance, gj, between cell pairs is often too large to permit visualization of unitary currents, which has necessitated the use of pharmacological agents to reduce the number of operational The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. channels (2). Furthermore, membrane capacity decreases frequency response well below that possible with excised patches, and ionic substitution studies, which depend on dialysis from patch electrodes, have been limited because of technical difficulties (3). Here we undertook the study of gap junction gating and permeation by recording from unapposed hemichannels. Although it is generally believed that unapposed hemichannels are closed in order to prevent loss of cytoplasmic solutes and entry of extracellular ions, exceptions include hemichannels formed by Cx46 (4, 5), Cx56 (6), and an unidentified connexin in fish horizontal cells (7). We expressed high levels of Cx46 in Xenopus oocytes and were able to record single hemichannel currents in both on-cell and excised, inside-out, and outsideout patch configurations. These hemichannels exhibit voltage gating consistent with the Vj dependence of hemichannels incorporated into cell-cell channels and in addition exhibit a novel voltage-gating mechanism that allows Cx46 hemichannels to open in the surface membrane of single cells. Multiple solution exchanges applied to the exposed face of excised patches indicated permeability to large ions and a significant preference for cations. These data demonstrate that unapposed hemichannels possess attributes characteristic of gap junction channels and establish the utility of the excised hemichannel preparation in probing mechanisms of gap junction voltage gating and permeation. MATERIALS AND METHODS Expression of Cx46 mRNA in Xenopus Oocytes. Cx46 DNA was cloned from rat genomic DNA using PCR amplification with primers corresponding to aminoand carboxyl-terminal sequences. Preparation of oocytes and synthesis of RNA have been described previously (8-10). Each oocyte was injected with 50 nl of an aqueous solution ofmRNA (2 mg/ml) together with DNA antisense to the endogenous XenCx38 (8 pmol/,Il) (11). We use the phosphorothioate antisense oligo 5'-GCT TTA GTA ATT CCC ATC CTG CCA TGT TTC-3', which is complementary to XenCx38 commencing at nt -5. Electrophysiological Recordings. Voltage clamp recordings of macroscopic currents from single Xenopus oocytes were obtained by utilizing a two-electrode voltage clamp. Both voltage-measuring and current-passing microelectrodes were filled with 1 M KCl. The oocytes were bathed in a solution containing 88 mM KC1, 2 mM MgCl2, 5 mM glucose, 10 mM Hepes, pH 7.6. Nominal Ca2+ in this solution was 5-10 ,uM as determined by a Moller Ca2+ ion-selective electrode. In all patch-clamp experiments, pipette solutions consisted of 100 mM KC1, 1 mM CaC12, 2 mM MgCl2, 5 mM EGTA, 10 mM Hepes, pH 7.6. Oocytes were bathed in a solution containing 88 mM NaCl, 1 mM KCl, 2 mM MgCl2, 5 mM glucose, 10 mM Hepes, pH 7.6. In experiments where relative permeabilities were determined, a flow chamber was designed with two separate compartments, one to hold the oocyte and Abbreviations: TEA, tetraethylammonium; TMA, tetramethylammonium.