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Advancing the pH hypothesis of negative feedback to photoreceptors: sources of protons and a role for bicarbonate in feedback

More than 40 years ago, it was found that retinal horizontal cells (HCs), second-order neurons of the outer retina, provide inhibitory feedback onto photoreceptors (PRs). This allows for lateral inhibition between PRs, through which HCs mediate edge detection and colour contrast. But the neurotransmitter or process behind this inhibitory feedback remains a mystery: although it is agreed that horizontal cells inhibit cones when they are depolarized, there is no consensus for the mechanisms of this feedback (Kramer & Davenport, 2015). Currently, there are three major hypotheses, each with supporting evidence (reviewed in Thoreson & Mangel, 2012; Kramer & Davenport, 2015): (i) the GABA hypothesis, in which depolarized horizontal cells release GABA onto cones, inhibiting them; (ii) the ephaptic hypothesis, in which horizontal cells change the extracellular voltage in the synaptic cleft, thereby modulating the transmembrane voltage gradient experienced by voltage-gated Ca2+ channels on cones; and/or (iii) the pH or proton hypothesis, in which depolarized horizontal cells release protons into the cleft to alter both the activation potential and peak currents of voltage-gated Ca2+ channels on cones. Each hypothesis has also generated some controversy (Thoreson & Mangel, 2012; Kramer & Davenport, 2015): GABA antagonists do not block feedback in many cases, and it is unclear how the ephaptic mechanism would account for observed changes in peak cone Ca2+ currents (ICa). A research article by Warren et al. (2016) in The Journal of Physiology addresses the main question mark which looms over the pH hypothesis: how do HCs change cleft pH? The proton hypothesis has gained much support in recent years. It is particularly attractive because it accounts for measured changes in PR ICa. Several lines of evidence also support pH-dependent feedback. A variety of pH buffers such as HEPES and Tris reduce feedback, as measured through both electrophysiology and Ca2+ imaging. Further evidence comes from a genetically encoded pH sensor localized to cone L-type Ca2+ channels at the synaptic cleft (Wang et al. 2014). In this model, light induces an alkalization of the cleft, in a time frame consistent with feedback from HCs to cones. Therefore, not only does the pH hypothesis account well for effects on cone ICa, but it has been illustrated with several different approaches, and is blockable. While other mechanisms may affect or contribute to feedback, a consensus is emerging that pH plays an important (if not central) role. The biggest hurdle that remains for the pH hypothesis is finding the source of protons. Warren et al. (2016) outline a number of potential sources for extracellular protons: (i) extracellular carbonic anhydrase (CA) can create protons and bicarbonate from CO2 and water, acidifying the cleft; (ii) vacuolar-type ATPases (v-ATPases) can acidify synaptic vesicles, which can acidify the synaptic cleft in turn, or they can be inserted onto membranes through vesicle fusion; and (iii) Na+/H+ exchangers (NHEs) are known to extrude protons from neurons. Although not examined by Warren et al. (2016), protons may also pass through other ion channels including hemichannels and epithelial Na+ channels (ENaCs) (Kramer & Davenport, 2015). As an alternative to proton release, cleft pH could be affected by changing the concentration of pH buffers. Several buffers (e.g. phosphate, bicarbonate) have been proposed (see references in Warren et al. 2016). To measure if CA affects feedback, Warren et al. (2016) measured whole-cell currents from voltage-clamped tiger salamander (Ambystoma tigrinum) cones, at various membrane potentials in a flat-mount preparation. First, they stimulated cones with spot stimuli; then, they recorded currents at light onand offset to measure feedback. When they applied a membrane-impermeant CA inhibitor, FC5-207a, they found changes in cone ICa consistent with an alkalization of the cleft. However, FC5-207a had no effect on feedback-induced changes in ICa. The authors concluded that CA contributes protons to the cleft, but is not the major source of protons involved in feedback. In other experiments, the authors then used a paired recording protocol, where they voltage-clamped HC membrane potentials to −90, −60, −30, or 0 mV and recorded from cones using a ramp protocol to measure both peak ICa and the membrane potential at 50% of peak ICa (V50). Values of peak ICa and V50 were recorded when HCs were relatively depolarized (i.e. at −60, −30 and 0 mV), and were then compared to peak ICa and V50 values at −90 mV. These differences in peak ICa and V50 were used as measures of feedback, since it is well-known that feedback involves changes in these two values. By voltage-clamping HCs, this set-up prevented feedback-induced reductions in HC membrane potential, which would in turn affect feedback to cones. Using this paired recording protocol, they measured the impact of v-ATPases and NHEs on cleft pH and feedback. The v-ATPase inhibitor bafilomycin A1 (BAF) seemed to increase initial cone currents upon depolarization and glutamate release. However, BAF did not affect feedback. Like CA, it seems that v-ATPases contribute protons to the cleft but are not central in mediating feedback. NHEs, however, seem to do both. Cariporide, a specific NHE antagonist, increases ICa, consistent with the notion that NHEs acidify the cleft. But both cariporide and Na+-free solutions significantly reduced feedback. Another NHE inhibitor, zoniporide, showed a similar trend that approached statistical significance. Warren et al. (2016) conclude that CA, v-ATPases, and NHEs all contribute protons to the cleft, yet NHEs are the dominant mechanism and are necessary for feedback. However, since NHEs are not voltage-dependent (whereas feedback is), NHEs cannot mediate feedback alone; the authors reason that after NHEs supply protons, another mechanism is responsible for feedback. The remainder of Warren et al.’s experiments implicated bicarbonate buffer in feedback. Bicarbonate has a high pKa, and would therefore increase cleft pH and reduce proton inhibition of PR Ca2+ channels. Extracellular bicarbonate was shown to be necessary for feedback-induced changes in V50 and peak ICa. However,