|Molecular Vision 2001;
Received 22 June 2001 | Accepted 13 November 2001 | Published 16 November 2001
Gating of retinal horizontal cell hemi gap junction channels by voltage, Ca2+, and retinoic acid
Douglas G. McMahon
Department of Physiology, University of Kentucky, Lexington, KY
Correspondence to: Douglas G. McMahon, Ph.D., Department of Physiology, University of Kentucky, Lexington, KY, 40536-0084; Phone: (859) 257-2345; FAX: (859) 323-1070; email: firstname.lastname@example.org
Purpose: Hemi gap junction (HGJ) channels, precursors of gap junctional channels, are functionally expressed in retinal horizontal cells where they may play roles in osmoeregulation and ephaptic regulation of synaptic feedback to photoreceptors. In this study we examined mechanisms of gating of these channels by transmembrane voltage, Ca2+ and retinoic acid (RA).
Methods: Experiments were performed on cultured bass horizontal cells using the conventional whole cell patch clamp configuration.
Results: HGJ currents in isolated bass horizontal cells, revealed by perfusion with Ca2+ free media, were opened by positive holding potentials and inhibited by negative holding potentials. These currents were also inhibited by external application of either Ca2+ or RA. Using a rapid perfusion system, the latency of 2 mM Ca2+ to begin channel closure was unmeasurably brief, whereas the latency for 30 mM RA action was 177±9 ms (mean±standard error of the mean). The total inhibition of HGJ channel currents by coapplication of 0.3 mM RA and 100 mM Ca2+ was less than the sum of inhibition by RA alone and Ca2+ alone suggesting that the actions of RA and Ca2+ were not independent. In the presence of 0.3 mM RA, the half maximal concentration for Ca2+ inhibition was increased from a control value of 192 mM to 375 mM without affecting maximal inhibition. Similarly, the half maximal concentration for RA inhibition was increased from a control value of 0.44 mM to 1.1 mM without affecting maximal inhibition in the presence of 100 mM Ca2+.
Conclusions: These results suggest that horizontal cell HGJ channels are closed by the normal negative resting potentials of these cells. Extracellular Ca2+ and the retinal neuromodulator RA also act to close HGJ channels through mechanisms or sites which are not independent.
Electrical transmission through gap junction channels plays an important role in the direct intercellular communication in the central nervous system as well as in the vertebrate retina [1,2]. Gap junctions are formed by connexins, gap junctional proteins which span two plasma membranes between adjacent cells. So far more than a dozen connexins have been cloned in mammals and functionally expressed in cell lines . The assembly of connexins into gap junctional channels may include two stages: connexin proteins first oligomerize into connexon hemichannels in the plasma membrane of individual cells and then the hemi gap junction (HGJ) channels in each of the neighboring cells align and dock in series to form gap junction channels between adjacent cells. Functional HGJ channels have been found in both native cells and cells transfected with connexins [4-16]. Their properties of gating and permeability are quite similar with those of the gap junctional channels: HGJ channels are membrane channels with a nonselective permeability that are gated by voltage and chemical uncouplers [4,8-10,13,16]. Therefore, HGJ channels are useful in the study the molecular structure and function of gap junction channels.
Retinal horizontal cells have been used extensively to study gap junctions and HGJ channels [4-6,10,16-25]. Functional HGJ channels of retinal horizontal cells are closed at normal extracellular Ca2+ concentrations but they can be opened by lowering Ca2+ experimentally [4,16]. HGJ channels are modulated by retinoic acid (RA), a light adapting substance in the vertebrate retina, through the action of a novel plasma membrane RARb/g-like binding site . In this study, we examined in detail the actions of voltage, Ca2+ and RA on HGJ channels of horizontal cells. The results show that HGJ channels are closed at negative potentials, that Ca2+ and RA act with different latencies and that RA interacts with Ca2+ in the gating of HGJ channels.
Dark adapted adult hybrid bass (Roccus chrysops and Roccus saxitalis) were euthanized in accordance with National Institutes of Health guidelines for animal use. Retinas were removed under dim red light and then incubated in L-15 media (GIBCO) containing 20 U/ml papain (Worthington Biochemical Corp., Lakewood, NJ) activated with 5.5 mM cysteine and 1.1 mM EDTA. The retinas were incubated in L-15/papain solution for 40 min followed by six changes of fresh L-15 media, and then dissociated by repeated passage through a serological pipette. Isolated cells were plated onto plastic 35 mm dishes containing fresh L-15 medium. Cultures were maintained at 17 °C and cells were used following 1 to 5 days in culture.
Recordings from horizontal cells were performed using the conventional whole cell patch clamp configuration. Patch pipettes were pulled from Corning 7052 glass (AM Systems Inc, Carlsborg, WA) and fire polished to a resistance of 3-4 MW. The pipette series resistance and capacitance were compensated by 80%. The offset potential between the pipette and bath solutions was zeroed prior to seal formation. The pipette solution contained 100 mM K-gluconate, 20 mM CsCl, 4 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 11 mM EGTA, 10 mM HEPES, and 10 mM TEA; the pH was adjusted to 7.5 with KOH. The normal bath solution contained 100 mM NaCl, 30 mM CsCl, 2 mM CaCl2, 2.5 mM KCl, 1 mM MgSO4, 1 mM Na-pyruvate, 10 mM HEPES, 16 mM glucose, 20 mM TEA, and 10 mM 4-AP; the pH was adjusted to 7.5 with NaOH. CaCl2 was omitted in Ca2+ free bath media. All-trans retinoic acid (at-RA; ICN Biomedicals, Inc., Costa Mesa, CA) was dissolved in DMSO as a stock solution of 100 mM.
Ultrafast solution exchange was achieved by using a theta tube mounted on a LSS 3100 piezoelectric driven actuator (Burleigh Instruments, Fishers, NY). Theta glass tubing, TGC 200-10, was purchased from Warner Instrument Corp. (Hamden, CT) and pulled to an outer tip diameter of 100-150 mm. To minimize the dead space along the application pipette, microfilament tubing MF 28G (World Precision Instruments, Inc., Sarasota, FL) was inserted up to the tip and then filled with molten wax almost to the tip. Movement of the theta tube and its two solution streams was automatically controlled by a voltage waveform consisting of ramps from 0 to 4 mV, 4 to 6 mV and 6 to 10 mV by Clampex 8 (Axon Instruments, Inc., Union City, CA) and then moving back in reverse order. Durations of ramp waveforms were 40 ms, 20 ms, and 40 ms respectively. In addition, slow application of drugs was used in some experiments. The solution was continuously delivered into the bath from a glass tube near the recorded cells at a rate of 3-4 ml/min.
Currents shown in the results were determined as the mean value of current amplitude for a 10 s interval recording period using Clampfit 8 (Axon Instruments). Normalized dose inhibition relationship data were fit with the Hill equation:
i / I = 1 - (An / [(IC50)n + An])
"I" and "i" are currents before and during application of the treatment (Ca2+ or RA), respectively. IC50 is the concentration giving a half maximal reduction. "A" represents a given Ca2+ or RA concentration and n is the Hill coefficient. Statistics on the data are presented as mean ± standard error of the mean.
Voltage, extracellular Ca2+ and retinoic acid (RA) have been shown to close HGJ channels of either native cells or cells transfected with recombinant connexins [4-16]. Figure 1 illustrates HGJ channels of bass horizontal cells were closed by negative holding potentials, Ca2+ and RA. The left trace of Figure 1A shows that prolonged depolarization to 30 mV from a holding potential of -60 mV evoked a slowly activating current in an H-2 type horizontal cell in the absence of external Ca2+. Upon repolarization to -60 mV, an inward tail current was observed with a quick inactivation to a steady state current near zero. When the cell was exposed to 2 mM Ca2+, both the outward current and inward tail current were almost completely abolished (right trace, Figure 1A) and they recovered upon washout (trace not shown). Consistent results were observed with 7 additional cells. Figure 1B shows the peak and steady state current-voltage relationships for HGJ channels constructed by subtracting tail current traces evoked by hyperpolarizing voltage clamp steps from a holding potential of 30 mV in the presence of 2 mM Ca2+ and in the absence of Ca2+ (n=4). The steady state current voltage relation was outwardly rectifying but the peak current-voltage relation was close to linear with a reversal potential of approximately 0 mV, indicating that the channels have a nonselective permeability. In Figure 1C, 0.3 mM at-RA reduced the amplitude of the peak outward current from 796 pA to 435 pA. On average, 0.3 mM reduced outward currents by 43% in 6 horizontal cells.
To examine the time course of Ca2+ action on HGJ channels, we performed experiments using a fast perfusion system having a 10 to 90% rise time of approximately 40 ms for whole cell recording. Before the experiments, the bathing solution in the dish was totally replaced by Ca2+ free solution. Then whole cell recordings were made, and cells depolarized to 30 mV from a holding potential of 0 mV with continuous perfusion of Ca2+ free solution. After the HGJ channel outward current was stable (3-5 min), the Ca2+ free perfusion solution was switched to 2 mM Ca2+ solution with a rapid transition. Figure 2B shows that 2 mM Ca2+ immediately inhibited HGJ channel current recorded from an H-1 horizontal cell. The upper trace (Figure 2A) is the pipette tip potential (a shift in holding current at voltage clamp mode) when switching perfusion solution from 10 mM TEA-Cl to 10 mM KCl after the experiment to illustrate the rise time of the perfusion transition. When the starting time of onset in the pipette tip potential was compared to the onset of HGJ channel closure, there was no measurable delay for Ca2+ reduction of HGJ channel current. Similar results were observed with other 3 cells.
Next, we determined the latency of retinoic acid action on HGJ channels in the same cells. The half maximal inhibition for RA of HGJ channel currents is 0.44 mM (Figure 3B). To obtain a maximal response a high concentration of at-RA (30 mM) was used. In the same cell that was shown in Figure 2B, the latency for RA action was 160 ms (Figure 2C). On average, the mean latency to the onset of channel closure for RA application was 177±9 ms (mean±standard error of the mean; n=4).
We then tested for interactions between Ca2+ and RA in modulating HGJ channels. Ca2+ action was examined in the presence and absence of RA. Figure 4A illustrates a recording from an H-2 type horizontal cell at a holding potential of 30 mV in Ca2+ free solution to give a total HGJ channel current of 484 pA. Application of 100 mM Ca2+ produced a 215 pA reduction in HGJ channel current. After washout, the perfusion solution was then switched to 0.3 mM RA which produced a 114 pA reduction of HGJ channel current. In the presence of 0.3 mM RA, application of 100 mM Ca2+ then produced 95 pA inhibition. The total inhibition of coapplication of RA and Ca2+ was 209 pA, whereas the sum of inhibition by Ca2+ alone and RA alone was 329 pA. These results indicate that the actions of RA and Ca2+ were not additive and therefore not independent. Similar results were obtained with 4 additional cells. Figure 4B shows the dose inhibition curves for Ca2+ on HGJ channel currents that were measured in the absence and in the presence of RA. In the presence of 0.3 mM RA, the half maximal concentration for Ca2+ was increased from a control value of 192 mM to 375 mM without affecting maximal inhibition. The Hill coefficient was increased from 1.1 to 1.6 in the presence of RA. The results indicate that RA decreased the apparent affinity of Ca2+ for HGJ channels without affecting the ability to fully close HGJ channels.
RA inhibition of HGJ channels was also modified by Ca2+. Figure 3A shows an example of Ca2+ reducing the action of RA on HGJ current in an H-2 type horizontal cell. Application of 0.3 mM at-RA produced a 249 pA inhibition in HGJ channel current in Ca2+ free solution. After washout, a 100 mM Ca2+ solution caused a 222 pA reduction of HGJ channel current. Coapplication of 100 mM Ca2+ and 0.3 mM at-RA produced a 272 pA reduction. The total inhibition (272 pA) by coapplication of Ca2+ and RA was less than the sum of inhibition (371 pA) by at-RA alone and Ca2+ alone again indicating that the actions of RA and Ca2+ were not additive. Consistent results were obtained with 3 additional cells. Figure 3B shows the dose inhibition curves for RA on HGJ channel currents that were measured in the absence and in the presence of Ca2+. In the presence of 100 mM Ca2+, the half maximal concentration for RA was increased from a control value of 0.44 mM to 1.1 mM without affecting maximal inhibition. The Hill coefficient was increased from 0.8 to 1.6 in the presence of Ca2+.
The findings of this study are that hybrid bass retinal horizontal cell HGJ channels are closed by negative resting potentials and Ca2+. In addition Ca2+, which like RA is thought to act directly on the external aspect of the HGJ channels to effect channel closure , interacts with RA in a non-additive manner. HGJ channel currents in isolated bass horizontal cells, revealed by perfusion with Ca2+ free media, were opened by positive holding potentials and inhibited by negative holding potentials in agreement with previously described HGJ channels in catfish and skate horizontal cells [4,5]. The inhibition at negative resting potentials typical for horizontal cells, combined with the inhibitory action of Ca2+ at normal extracellular concentrations, suggests that there would be few HGJ channels open on bass horizontal cells in situ.
The rapid time course of Ca2+ closure of HGJ channels suggests an extracellular site of action, in agreement with previous reports of HGJ channel modulation by Ca2+ . Although we cannot rule out an involvement of an increase in intracellular Ca2+ during prolonged extracellular application of Ca2+, the immediate nature of Ca2+'s initial action strongly suggests that extracellular Ca2+ acted directly on the connexins to reduce HGJ channel activity independent of any cytoplasmic factors. Also, the voltage independent inhibition by Ca2+ of fish horizontal cell HGJ channel activity suggests that the Ca2+ binding site is unlikely to be within the channel pore . In connexin46, the Ca2+ gating site of HGJ channels is indeed located extracellularly . So far, it is unclear what kinds of connexins are expressed by bass horizontal cells. The possible connexins may be cx26, cx34.7, cx35, cx43, cx27.5, cx44.1 and cx55.5, which were cloned from fish retinas or enriched preparations of fish horizontal cells [25-29]. Some of them have been shown to have functional HGJ channels expressed in Xenopus oocytes which are sensitive to Ca2+ and quinine .
We found that in contrast to Ca2+, RA which also closes HGJ channels with an extracellular site of action , exhibited an onset latency of 150-200 ms. Since we previously ruled out that RA acts through pH, lipophilic action, or second messengers , the latency of RA action might be produced by an interaction of RA with RA binding proteins, which then interact with the HGJ channel, or the binding of RA to the HGJ channel itself may be a more complex process than Ca2+-binding.
RA and Ca2+ also interacted in their gating of HGJ channel currents of horizontal cells. RA decreased the apparent affinity of Ca2+ for HGJ channels without inhibiting maximum closure, whereas Ca2+ had an identified effect on RA action. These interactions are consistent with competitive inhibition between Ca2+ and RA in gating HGJ channels. Additional binding experiments will be necessary to ascertain this with certainty. Whether RA's interaction with Ca2+ takes place directly at a common binding site or indirectly cannot be rigorously concluded from the present data. At the very least, RA must interact with a site other than the Ca2+ site on HGJ channels since the HGJ channel Ca2+ site is not accessible in the fully assembled state, yet RA can modulate intact gap junctions as well . In this regard it is interesting to note that the half maximal concentration for RA uncoupling of gap junctions is about 5-fold greater than for HGJ channel modulation . Thus, channel assembly into junction complexes may mask or modify a high affinity RA site on connexins.
Previously, the unique physiological function of membrane HGJ channels was thought to be to assemble into gap junction channels between two apposing cells. Recently, other functions of HGJ channels, as membrane ion channels in the non-junctional regions of individual cells, have been proposed. HGJ channels were found to be mediate cell volume regulation, transmembrane fluxes of a nucleotide, and signal transmission [30-32]. For example, in response to a change in the extracellular physiological Ca2+ concentration, cell volume significantly and reversibly increased in cells expressing cloned connexin channels as well as fibroblasts and endothelial cells . In the vertebrate retina, RA, with other endogenous uncouplers, such as dopamine and nitric oxide, may modulate HGJ channels to maintain cell volume. Most recently, evidence has been presented that Cx26 is expressed in carp retinal horizontal cells, and that the expression is restricted to the terminal dendrites apposing photoreceptors . Further experiments have suggested that HGJ channels on carp horizontal cells mediate negative feedback from horizontal cells to photoreceptors by an ephaptic mechanism . The gating of HGJ channels by voltage, Ca2+ levels and retinally released RA will shape their participation in such a mechanism and thus may influence the function of outer retinal neural network [31,33,34].
We thank Dr. C. G. Parsons for sharing his experience in using the rapid perfusion system. This work was supported by National Eye Institute Grant R01EY09256 to DGM.
1. Bruzzone R, Ressot C. Connexins, gap junctions and cell-cell signalling in the nervous system. Eur J Neurosci 1997; 9:1-6.
2. Cook JE, Becker DL. Gap junctions in the vertebrate retina. Microsc Res Tech 1995; 31:408-19.
3. Kumar NM, Gilula NB. Molecular biology and genetics of gap junction channels. Semin Cell Biol 1992; 3:3-16.
4. DeVries SH, Schwartz EA. Hemi-gap-junction channels in solitary horizontal cells of the catfish retina. J Physiol 1992; 445:201-30.
5. Malchow RP, Qian H, Ripps H. Evidence for hemi-gap junctional channels in isolated horizontal cells of the skate retina. J Neurosci Res 1993; 35:237-45.
6. Malchow RP, Qian H, Ripps H. A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina. J Gen Physiol 1994; 104:1039-55.
7. Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG. Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol 1996; 134:1019-30.
8. Ebihara L, Berthoud VM, Beyer EC. Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys J 1995; 68:1796-803
9. Trexler EB, Bennett MV, Bargiello TA, Verselis VK. Voltage gating and permeation in a gap junction hemichannel. Proc Natl Acad Sci U S A 1996; 93:5836-41.
10. Dixon DB, Takahashi K, Bieda M, Copenhagen DR. Quinine, intracellular pH and modulation of hemi-gap junctions in catfish horizontal cells. Vision Res 1996; 36:3925-31.
11. Castro C, Gomez-Hernandez JM, Silander K, Barrio LC. Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations. J Neurosci 1999; 19:3752-60.
12. Trexler EB, Bukauskas FF, Bennett MV, Bargiello TA, Verselis VK. Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation. J Gen Physiol 1999; 113:721-42.
13. White TW, Deans MR, O'Brien J, Al-Ubaidi MR, Goodenough DA, Ripps H, Bruzzone R. Functional characteristics of skate connexin35, a member of the gamma subfamily of connexins expressed in the vertebrate retina. Eur J Neurosci 1999; 11:1883-90.
14. Pfahnl A, Dahl G. Gating of cx46 gap junction hemichannels by calcium and voltage. Pflugers Arch 1999; 437:345-53.
15. Verselis VK, Trexler EB, Bukauskas FF. Connexin hemichannels and cell-cell channels: comparison of properties. Braz J Med Biol Res 2000; 33:379-89.
16. Zhang DQ, McMahon DG. Direct gating by retinoic acid of retinal electrical synapses. Proc Natl Acad Sci U S A 2000; 97:14754-9.
17. Lasater EM, Dowling JE. Dopamine decreases conductance of the electrical junctions between cultured retinal horizontal cells. Proc Natl Acad Sci U S A 1985; 82:3025-9.
18. Lasater EM. Retinal horizontal cell gap junctional conductance is modulated by dopamine through a cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A 1987; 84:7319-23.
19. McMahon DG. Modulation of electrical synaptic transmission in zebrafish retinal horizontal cells. J Neurosci 1994; 14:1722-34.
20. Mills SL, Massey SC. Distribution and coverage of A- and B-type horizontal cells stained with Neurobiotin in the rabbit retina. Vis Neurosci 1994; 11:549-60.
21. McMahon DG, Mattson MP. Horizontal cell electrical coupling in the giant danio: synaptic modulation by dopamine and synaptic maintenance by calcium. Brain Res 1996; 718:89-96.
22. Lu C, McMahon DG. Modulation of hybrid bass retinal gap junctional channel gating by nitric oxide. J Physiol 1997; 499:689-99.
23. Lu C, Zhang DQ, McMahon DG. Electrical coupling of retinal horizontal cells mediated by distinct voltage-independent junctions. Vis Neurosci 1999; 16:811-8.
24. Pottek M, Weiler R. Light-adaptive effects of retinoic acid on receptive field properties of retinal horizontal cells. Eur J Neurosci 2000; 12:437-45.
25. Janssen-Bienhold U, Schultz K, Gellhaus A, Schmidt P, Ammermuller J, Weiler R. Identification and localization of connexin26 within the photoreceptor-horizontal cell synaptic complex. Vis Neurosci 2001; 18:169-78.
26. O'Brien J, al-Ubaidi MR, Ripps H. Connexin 35: a gap-junctional protein expressed preferentially in the skate retina. Mol Biol Cell 1996; 7:233-43.
27. O'Brien J, Bruzzone R, White TW, Al-Ubaidi MR, Ripps H. Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. J Neurosci 1998; 18:7625-37.
28. Wagner TL, Beyer EC, McMahon DG. Cloning and functional expression of a novel gap junction channel from the retina of Danio aquipinnatus. Vis Neurosci 1998; 15:1137-44.
29. Dermietzel R, Kremer M, Paputsoglu G, Stang A, Skerrett IM, Gomes D, Srinivas M, Janssen-Bienhold U, Weiler R, Nicholson BJ, Bruzzone R, Spray DC. Molecular and functional diversity of neural connexins in the retina. J Neurosci 2000; 20:8331-43.
30. Quist AP, Rhee SK, Lin H, Lal R. Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 2000; 148:1063-74.
31. Kamermans M, Fahrenfort I, Schultz K, Janssen-Bienhold U, Sjoerdsma T, Weiler R. Hemichannel-mediated inhibition in the outer retina. Science 2001; 292:1178-80.
32. Bruzzone S, Guida L, Zocchi E, Franco L, De Flora A. Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. FASEB J 2001; 15:10-2.
33. McCaffery P, Mey J, Drager UC. Light-mediated retinoic acid production. Proc Natl Acad Sci U S A 1996; 93:12570-4.
34. Vellani V, Reynolds AM, McNaughton PA. Modulation of the synaptic Ca2+ current in salamander photoreceptors by polyunsaturated fatty acids and retinoids. J Physiol 2000; 529:333-44.