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Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, UIC College of Medicine, Chicago, Illinois 60612
Signal transmission through gap-junctional channels serves to synchronize and regulate a broad range of cellular activities in tissues throughout the body (1). The structural proteins that constitute gap-junctional channels are the connexins, a multigene family of homologous proteins, whose members are identified by their predicted molecular mass in kilodaltons (e.g., Cx35). Six connexin polypeptides oligomerize to form a membrane hemichannel, or connexon, which can dock with the connexons from adjacent cells to form an aqueous pore (d 
16 Å) which allows the cell-to-cell diffusion of ions, second-messenger molecules, and metabolites having a molecular mass 
1 kDa. The delivery and insertion of connexin subunits in the plasma membrane and their assembly into connexons represent important stages in the formation of gap junctions. It is not surprising, therefore, that the gap-junctional channels of electrically coupled cells and the hemichannels formed by their connexon precursors often exhibit similar regulatory mechanisms and gating properties (2).
Because the Xenopus oocyte has the capacity to efficiently translate foreign genetic information and to assemble and insert oligomeric channel complexes into its plasma membrane, it has become a widely used expression system for the functional analysis of gap-junctional proteins. Oocytes expressing connexin subunits, and brought into contact after removal of the follicular and vitelline membranes, typically form gap-junction channels, affording the opportunity for electrophysiological and pharmacological study. In addition, there is growing interest in the functional properties of the hemichannels formed in isolated neurons (3,4) and in single oocytes (5) expressing various gap-junctional proteins. The presence of non-junctional hemichannels is readily demonstrated electrophysiologically. Prolonged membrane depolarization results in the development of a time- and voltage-dependent outward current attributable to the opening of hemi-gap-junctional channels. However, the same voltage protocol brings into play a Na+-dependent current of similar time course, but opposite polarity (6). Although this slow inward current is clearly different from the transient Na+ current of neurons and muscle cells, which inactivates rapidly during depolarization and is abolished by tetrodotoxin at concentrations about 1000 times lower than that required to block the sustained inward current (7), it tends to reduce the apparent amplitude of the hemichannel current or greatly restrict the voltage range over which it can be reliably recorded. The interaction between these two currents can be particularly troublesome when attempting a quantitative pharmacological analysis of hemichannel activity. Most agents that modulate gap-junctional communication produce a reduction in channel conductance, and thus low-amplitude hemichannel currents are not suitable for obtaining dose-response data over a significant concentration range.
In the present study, we used the two-electrode voltage-clamp technique to examine the interaction between the sodium current and the hemichannel currents mediated by the endogenous connexin (Cx38) of Xenopus oocytes. As illustrated in Figure 1, the large inward sodium currents elicited in the normal modified Barth’s (MB) solution (Fig. 1A) obscured the weaker hemichannel currents. However, when we removed extracellular sodium (choline substitution) the presence of the underlying hemichannel currents was revealed (Fig. 1B). Although the small hemicurrents recorded from some oocytes may be attributable to a low level of Cx38 expression, in most cases the sodium current either completely masked hemichannel activity (as in Fig. 1A) or reduced its magnitude to such an extent as to preclude pharmacological studies.
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With the full magnitude of the hemichannel currents exposed, either by replacing sodium with choline in the MB solution or by the addition of quinine, we could conduct pharmacological studies of agents that are known to modulate gap-junctional conductances in coupled neurons and other cell types. All-trans retinoic acid (RA), a metabolite of vitamin A, acting through a plasma membrane retinoic acid-binding protein, has been shown to reduce electrical coupling between horizontal cells both in situ and between cell pairs in culture (10). A similar action was observed in our recordings of Cx38-mediated hemichannel recordings from Xenopus oocytes superfused with a Na-free solution supplemented with various concentrations of RA (Fig. 1E); the IC50 of approximately 2 µM is comparable to that obtained with coupled horizontal cells in culture (10). Figure 1F illustrates the results obtained with carbenoxolone, applied to an MB solution containing 200 µM quinine. Carbenoxolone is one of the many derivatives of glycyrrhetinic acid, a saponin obtained from the licorice root Glycyrrhizia glabra, that has been shown to be a potent inhibitor of gap-junctional communication in various cell types (11). Figure 1F shows that the drug blocked Cx38 hemichannel activity with an IC50 of approximately 34 µM.
In sum, our study describes some of the membrane properties of Xenopus oocytes in response to prolonged depolarizing voltage steps, and illustrates the interaction between an inward sodium current and the hemichannel current mediated by the endogenous gap-junctional protein, Cx38. Blocking the Na+ current enhanced the recorded hemichannel currents, and enabled us to show that the chemical gating of Cx38 hemichannels corresponds in many respects to that observed with fully formed gap-junctional channels in other systems. The use of this "modified" oocyte hemichannel preparation should prove useful for developing response profiles or "signatures" with which to distinguish the properties of one connexin from another, and for determining the modulatory effects of pharmacological agents that affect the gating of gap-junctional channels.
These studies were conducted at the Marine Biological Laboratory, Woods Hole, Massachusetts, and supported in part by grants from the National Eye Institute (EY-06516, EY- 12028, and EY-01792); an unrestricted award to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc.; a Career Development Award from RPB (HQ); and an Award from the Alcon Research Institute (HR).
Literature Cited
This article has been cited by other articles:
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  M. C. Puljung, V. M. Berthoud, E. C. Beyer, and D. A. Hanck Polyvalent Cations Constitute the Voltage Gating Particle in Human Connexin37 Hemichannels J. Gen. Physiol., October 25, 2004; 124(5): 587 - 603. [Abstract] [Full Text] [PDF]
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+30 mV) only inward currents are seen. The MB solution contained [in mM]: NaCl [88], KCl [1], NaHCO3 [2.4], N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) [15], Ca(NO3)2 [0.33], CaCl2 [0.41], and MgSO4 [0.82]; 10 mg/liter gentamycin was added, and the solution titrated with NaOH to pH7.4. (B) After replacing the bath solution with Na+-free (choline substitution) MB solution, voltage steps