Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse

تعليق: مضخات البروتون في الشبكية وعلاقتها بكهربيتها وأيضاً بالجلوتاميت وهوالإكسيتوتوكسين Excitotoxin. وأيضاً نتسائل هل الأميلوريد والأسيتازولاميد = دياموكس/سيديمكس والنيكل والكوبالت لهم تأثير سلبي على النظر. يجب مراجعة المنشورات عن الأعراض الجانبية البصرية للدياموكس والأميلوريد وهل هي مشهورة أم نادرة؟ ودرجة التحذير منها وهل لها علاقة بمثبطات مضخات البروتون. مع مملاحظة أن تغيير بسيط في الأس الهيدروجيني يؤدي لتغيير في الكهرباء ربما كبير A change of extracellular pH by 0.1 units yields a shift in calcium channel gating of ∼1 mV (Barnes and Bui, 1991). وأيضاً ربما ذو صلة عدم إختفاء تأثير مركب الكايييين KAine على الشبكية بسهولة حتى بعد التعرض له بكميات بسيطة ولفترات محدودة فهل هذا نفس أثر الأوميبراز وعائلته على الشبكية. Washout of the drug could not be achieved in the time course of these experiments. This was not surprising because the effects of a short exposure to micromolar concentrations of kainate have been shown to persist in cells of the retina for up to 10 min (Baldridge, 1996). --احمد شوقي محمدين 16:32، 3 نوفمبر 2016 (ت ع م)

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals.

We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones

J Neurosci. 2005 Apr 20;25(16):4108-17.

DOI: 10.1523/JNEUROSCI.5253-04.2005

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Vessey JP1, Stratis AK, Daniels BA, Da Silva N, Jonz MG, Lalonde MR, Baldridge WH, Barnes S.

Abstract

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

Introduction

At the output synapse of cone photoreceptors, horizontal cells (HCs) contribute to the surround receptive field of cones by sending illumination-sensitive inhibitory feedback to the presynaptic cones (Baylor et al., 1971). Inhibitory feedback underlies the formation of center-surround antagonistic receptive fields, which support edge detection, create color opponency, and enhance contrast (Wu, 1992; Burkhardt, 1993; Twig et al., 2003). How this inhibition is generated is not yet understood.

Of several mechanisms proposed (Kamermans and Spekreijse, 1999), the proton model for inhibitory feedback at the cone synapse is attractive for several reasons. First, protons are released along with glutamate from cone synaptic vesicles and inhibit voltage-gated Ca channels (DeVries, 2001). Proton pumps acidify synaptic vesicles to establish a driving force for glutamate uptake, resulting in vesicles that are ∼1.5 pH units more acidic than cytoplasm (Liu and Edwards, 1997). During fusion with the presynaptic membrane, both glutamate and protons are released into the cleft. HCs also extrude protons as a result of normal cell metabolism (Haugh-Scheidt and Ripps, 1998). Second, proton-mediated inhibition of cone calcium channels has been well documented (Barnes and Bui, 1991; Barnes et al., 1993; DeVries, 2001). Protons inhibit Ca channels in two ways. They reduce the conductance of Ca channels by binding to a site in or near the channel pore, interfering with the interaction between charged amino acids and the ions passing through the pore (Prod'hom et al., 1987; Chen et al., 1996). Protons also neutralize fixed negative surface charges on the plasma membrane, shifting Ca channel open probability to more positive voltages (Krafte and Kass, 1988; Klockner and Isenberg, 1994). In an acidic environment, increased membrane depolarization is required to overcome proton inhibition and open the channel. A change of extracellular pH by 0.1 units yields a shift in calcium channel gating of ∼1 mV (Barnes and Bui, 1991).

The negative shift in Ca channel activation caused by surround illumination (Verweij et al., 1996; Hirasawa and Kaneko, 2003) could be the result of a pH increase in the synaptic cleft. By increasing the proton buffering capacity of the bathing solution to reduce changes of extracellular pH, feedback-induced responses of both cones and HCs are attenuated (Hirasawa and Kaneko, 2003). Although multiple sources of protons exist, no precise mechanism by which HCs modulate pH in a voltage-dependent manner has been identified. In this paper, we show that, not only is the feedback signal sensitive to increased proton buffering, it is sensitive to amiloride, carbonic anhydrase inhibitors, and the divalent cations nickel and cobalt. A proton-conducting ion channel in the HC dendrites could provide a suitable pH-regulating influence in the synaptic cleft because HC hyperpolarization would increase the inward driving force on protons, increasing cleft pH, and depolarization would reduce the inward driving force, leaving the cleft more acidic.

methazolamide, a carbonic anhydrase inhibitor

What membrane mechanism might HCs use to regulate cleft pH in a voltage-dependent manner? If the data presented so far suggest that a decrease in cleft proton concentration accompanies HC hyperpolarization, this might be accomplished by diffusion of protons down their electrochemical gradient, in which case, simple depletion of an inward proton gradient across the cell membrane or interference with proton conductance mechanisms should reduce that regulatory ability. To begin examining this, we used methazolamide, a carbonic anhydrase inhibitor, to acidify the cytoplasm of HCs. Inhibition of carbonic anhydrase should cause proton concentrations to rise in the cell body, an effect found previously in cultured astrocytes (Chow et al., 1991, 1992). By acidifying the intracellular compartment, the proton driving force would be decreased, and, when hyperpolarized, HCs ought not be able to alkalinize the synaptic cleft via increased proton influx.

amiloride

Similar effects on feedback were found with amiloride, a known blocker of sodium/proton exchangers (Aickin and Thomas, 1977) and epithelial Na channels (ENaCs) (Garty and Benos, 1988).

............ When tested in the isolated goldfish retina, amiloride (200 μm) abolished rollback in the H1 horizontal cell light response (Fig. 7B). Before application of amiloride, H1 cells displayed 11 ± 2% rollback. In the presence of amiloride, rollback was significantly reduced or eliminated (p < 0.05; four retinas), and instead a6 ± 7% increase in the hyperpolarizing response was found. In three of the retinas, recordings were possible after washout of amiloride and in these cells rollback recovered (11 ± 4%). The average hyperpolarizing response amplitude under control conditions was -23 ± 4 mV, in the presence of amiloride was -16 ± 4 mV, and after wash was -21 ± 3 mV (means ± SEM). Although the mean values indicate a trend to reduced light responses in amiloride, the differences are not significant

النيكل والكوبالت

A selective, inhibitory action of Ni and Co on surround-induced feedback signals in the retina has been known for many years (Thoreson and Burkhardt, 1990; Vigh and Witkovsky, 1999), an effect consistent with the effects of these divalents shown here.

.......... Amiloride-sensitive ENaCs present a reasonable target because these channels are known to conduct protons and are blocked by the divalent cations Co2+ and Ni2+ (Hille, 2001; Sheng et al., 2004).

Discussion

Synaptic feedback in the outer retina could be encoded by changes in extracellular pH

We describe a near complete reduction in feedback from HCs to cones when pH buffering power is increased. In H1 cells, feedback is seen as a rollback of the hyperpolarizing response to light. Increasing bath pH buffering reduced, eliminated, or even reversed this depolarizing trajectory. In H2 cells, a depolarizing response to red light arises from the negative feedback of H1 cells to green cones. When H1 cells hyperpolarize to red light, this signal is transferred via sign-inverting feedback to green cones, which subsequently feed forward to H2 HCs and produce depolarization. Here again, increasing pH buffering eliminated the red-induced depolarization as well as increasing the hyperpolarization caused by green light. A hypothetical explanation for these effects is that HCs regulate pH in the synaptic cleft and that changes in cleft pH carry a critical element of the feedback signal to cones that can be quenched by strong pH buffers.

To frame this hypothesis more precisely, we tested drugs known to depolarize or hyperpolarize HCs and found remarkable actions on presynaptic cone synaptic terminals. Kainate, which typically depolarizes neurons and increases [Ca2+]i, dramatically reduced the Ca signals in cones, whereas CNQX, which is known to hyperpolarize HCs and is typically associated with a reduction in Ca signals, unexpectedly produced a marked increase in the depolarization-induced Ca signals of cone synaptic terminals. The direct effects of these drugs on HC Ca signals were difficult to detect. In fact, kainate-induced increases of [Ca2+]i in HCs were observed only sporadically because, in general, HCs did not take up the calcium indicator. This fact also precluded direct observation of a hyperpolarization-induced reduction of Ca signal in response to CNQX.

HC membrane potential drives Ca signal modulation in presynaptic cones

We showed that kainate and CNQX push the inhibitory feedback signal in opposing directions, presumably by shifting the presynaptic cone Ca channel activation curve in the positive or negative direction, respectively (Hirasawa and Kaneko, 2003). These drugs may achieve presynaptic modulation by acting on glutamate receptors found on dendrites of HCs that project into the synaptic invaginations of cone pedicles. The absence of AMPA or kainate receptor subunits on photoreceptors has been shown previously (Tachibana and Kaneko, 1988; Haverkamp et al., 2001a,b; Hirasawa and Kaneko, 2003), and we note that a Cl- conductance increase coupled to glutamate uptake (Sarantis et al., 1988; Arriza et al., 1997) is antagonized by kainate (Eliasof and Werblin, 1993; Eliasof et al., 1998). The lack of any detectable responses in the synaptic terminal layer of the zebrafish retina to the application of kainate in the present studies is consistent with the absence of AMPA receptors in photoreceptors. Kainate, by activating AMPA receptors, depolarizes HCs in the retinal slice. Because the imaging experiments are performed on light-adapted retinas, the HCs should be hyperpolarized and the depolarizing response is quite large. By depolarizing HCs, the feedback inhibition they send to the presynaptic cone Ca channels is enhanced. Conversely, CNQX antagonizes AMPA receptors, blocking the effects of glutamate released by the cones. This hyperpolarizes HCs further and reduces their inhibitory feedback to cone Ca channels. When revealed with a depolarizing (K+) stimulus, the calcium signal in the synaptic terminals was suppressed by kainate and enhanced by CNQX, providing evidence of modulation of the inhibitory feedback signal.

Inhibitory feedback from HCs to cones is sensitive to increased pH buffering and membrane proton flux

We showed that several specific treatments interfered with the presynaptic Ca signal changes induced by kainate and CNQX. When bicarbonate was added or the concentration of HEPES was increased, extracellular pH within the retinal slice, including the invaginating synapses, should have been strongly clamped. Were feedback encoded by extracellular protons, this would reduce changes in cleft pH in response to the application of either kainate or CNQX. Increased HEPES abolished the CNQX-mediated effect and attenuated that of kainate, suggesting that kainate and CNQX lead to shifts in cleft pH that alter the gating kinetics of the presynaptic cone Ca channels.

In addition to the sensitivity to increased pH buffering, the inhibitory feedback signal was also found to be sensitive to a reduction in proton driving force. Methazolamide, a carbonic anhydrase inhibitor, blocked the effects of CNQX but not kainate. By disrupting the ability of HCs to regulate internal pH (pHi), it is expected that pHi will decrease (as a result of continued cellular metabolism). CNQX, by hyperpolarizing HCs, increases the inward proton driving force and alkalinizes the synaptic cleft, an effect blocked by the accumulation of intracellular protons as a result of methazolamide application. Conversely, by depolarizing HCs, kainate decreases proton driving force permitting the cleft to stay acidified. We postulate that, for this reason, methazolamide was without effect on the cone [Ca2+]i response elicited with HC depolarization.

Presynaptic Ca channels are strongly sensitive to pH

This work suggests that the HC light responses recorded in intact goldfish retina have the same sensitivity to increases in pH buffering as the Ca signals recorded optically in retinal slices. What are the most likely targets of protons at the cleft?

The effect of protons on Ca channel gating has been examined (Barnes and Bui, 1991; Barnes et al., 1993; DeVries, 2001). In the current studies, acidic conditions reduced depolarization-induced calcium signals, whereas basic conditions increased the calcium signals. After testing a range of pH values, it was possible to estimate the pH value that kainate and CNQX induce in the cleft. Kainate at 50 μm may acidify the cleft to approximately pH 6.9, whereas 50 μm CNQX may alkalinize the cleft to approximately pH 7.9. A change of 0.1 pH units shifts the activation curve of cone Ca channels by ∼1 mV (Barnes and Bui, 1991), and surround illumination shifts the activation curve of the Ca channels negatively by ∼7.5 mV (Verweij et al., 1996). Therefore, modulation of cleft pH by kainate and CNQX appears consistent with a proton-mediated mechanism of inhibitory feedback on Ca channels.

We note that we may have overestimated the pH changes responsible for presynaptic Ca signaling in feedback: recordings of isolated cones and HCs in slices show that Ca channel activity is sufficiently suppressed at pH 6.9 to eliminate synaptic transmission at this synapse (Barnes and Bui, 1991; Barnes et al., 1993).

Horizontal cells could modulate cleft pH via an amiloride-sensitive proton channel

The extracellular compartment of the outer retina is known to undergo a light-induced alkalinization (Borgula et al., 1989; Oakley and Wen, 1989; Yamamoto et al., 1992). These measurements are consistent with the general principal that depolarization increases metabolic load on cells, and this leads to an increase in the production and extrusion of protons. Two reports suggest that glutamate reduces proton efflux from isolated HCs (Dixon et al., 1993; Molina et al., 2004), a finding in contrast to what this proton model of feedback requires. This efflux is coupled to Ca-ATPase activity and is observed when cells are bathed in a strongly pH-buffered environment (a proton sink). With weaker proton buffering and a restricted extracellular volume, conditions of elevated cleft proton concentration would lead to proton influx via proton-permeable channels. There are multiple sources of cleft protons associated with cell metabolism, and protons are released from cones along with vesicular glutamate (DeVries, 2001).

Estimates put the volume of the invaginating synaptic cleft in the range of 3 × 10-18 L (Raviola and Gilula, 1975), a volume in which approximately two protons give rise to a pH of 6. Because it has been shown that vesicular release produces a significant source of protons in the cleft at ribbon synapses in cones (DeVries, 2001) and bipolar cells (Palmer et al., 2003), given the partial protonation of vesicular glutamate molecules (intravesicular pH ∼5.7; pKa of glutamate carboxyl side chain of 4.4) and a dark release rate estimated at 400 μm/s (Roska et al., 1998) into an ∼3 ×10-18 L volume, the flux of protons to maintain steady-state pH could be in the range of ∼40 protons per second per cleft. This flux represents an extremely small current (0.006 fA) to be accommodated by a proton conductance. To alkalinize the cleft during HC hyperpolarization, slightly larger fluxes would need to be accommodated.

Amiloride-sensitive channels, such as ENaCs, would be suitable candidates for the HC pH-regulating mechanism. Amiloride is known to inhibit some ENaCs (Garty and Palmer, 1997), acid sensing ion channels (Waldmann et al., 1997), transient receptor potential channels (Inoue et al., 2001; Vulcu et al., 2004), and glutamate-gated channels (Manev et al., 1990). Divalent cations have been shown to block inhibitory feedback from HCs, and no conclusive explanation for this has been put forth (Thoreson and Burkhardt, 1990; Vigh and Witkovsky, 1999; Fahrenfort et al., 2004). Divalent cations block ENaC currents (Sheng et al., 2002). Our imaging of Ca signals demonstrated sensitivity to both divalent cations and amiloride. We also showed that amiloride blocks the rollback in H1 cells and reduces whole-cell currents in isolated HCs. ENaCs also display a high proton conductance (Hille, 2001), necessary for a role in extracellular pH regulation. ENaC subunits have been shown to be present in the retina with molecular and immunological techniques (Mirshahi et al., 1999; Golestaneh et al., 2000), and the α-subunit of the ENaC family is expressed in the outer plexiform layer (Brockway et al., 2002). Although the outer retina presents a diversity of targets for amiloride (and the other agents tested), these data are consistent with the notion that an HC proton conductance underlies generation of the voltage-dependent feedback response.

PMID: 15843613

رابط

https://www.ncbi.nlm.nih.gov/pubmed/15843613

http://jneurosci.org/content/25/16/4108.long