Sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in Ambystoma tigrinum retina

Abstract

Lateral-inhibitory feedback from horizontal cells (HCs) to photoreceptors involves changes in synaptic cleft pH accompanying light-evoked changes in HC membrane potential. We analysed HC to cone feedback by studying surround-evoked light responses of cones and by obtaining paired whole cell recordings from cones and HCs in salamander retina. We tested three potential sources for synaptic cleft protons: (1) generation by extracellular carbonic anhydrase (CA), (2) release from acidic synaptic vesicles and (3) Na /H exchangers (NHEs). Neither antagonizing extracellular CA nor blocking loading of protons into synaptic vesicles eliminated feedback. However, feedback was eliminated when extracellular Na was replaced with choline and significantly reduced by an NHE inhibitor, cariporide. Depriving NHEs of intracellular protons by buffering HC cytosol with a pH 9.2 pipette solution eliminated feedback, whereas alkalinizing the cone cytosol did not, suggesting that HCs are a major source for protons in feedback. We also examined mechanisms for changing synaptic cleft pH in response to changes in HC membrane potential. Increasing the trans-membrane proton gradient by lowering the extracellular pH from 7.8 to 7.4 to 7.1 strengthened feedback. While maintaining constant extracellular pH with 1 mm HEPES, removal of bicarbonate abolished feedback. Elevating intracellular bicarbonate levels within HCs prevented this loss of feedback. A bicarbonate transport inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), also blocked feedback. Together, these results suggest that NHEs are the primary source of extracellular protons in HC feedback but that changes in cleft pH accompanying changes in HC membrane voltage also require bicarbonate flux across the HC membrane.

Membrane‐associated guanylate kinase scaffolds organize a horizontal cell synaptic complex restricted to invaginating contacts with photoreceptors

“…Strong pH buffering and alkalinization of the extracellular medium enhance photoreceptor Ca 2+ current and prevent further enhancement caused by surround illumination that hyperpolarizes HCs, indicating that feedback has been blocked (Hirasawa and Kaneko, ; Vessey et al, ; Cadetti and Thoreson, ). Recent studies propose that protons responsible for acidifying the synaptic cleft come from HCs via an Na + :H + exchanger (Warren et al, ) but that regulation of extracellular buffering by bicarbonate is responsible for the modulation of pH (Liu et al, ; Warren et al, ). An alternative hypothesis is that changes in phosphate buffering developed via ATP release through pannexin 1 channels and extracellular hydrolysis of ATP are responsible for the change in cleft pH (Vroman et al, ).…”

Transretinal ERG in Studying Mouse Rod Phototransduction: Comparison With Local ERG Across the Rod Outer Segments

“…10,22 HEPES, present in both solutions, also blocks the pHsensitive feedback form horizontal cell to cone photoreceptors although its main purpose in our study was pH buffering. 23,24 All chemicals were purchased from SigmaAldrich (Espoo, Finland). The perfusion was connected to the signal ground with a 4.7-lF capacitor to filter highfrequency noise.…”

“…The bicarbonate buffered situation resembles more closely to the situation in vivo because bicarbonate regulates many retinal functions, including the intracellular pH, 41,42 the guanylate cyclase activity, 43 and the pH of the synaptic region known to modulate the calcium currents in photoreceptors. 24 Nevertheless, the mechanisms responsible for generating this component remain unclear but it most likely originates in the rod inner segment and the synaptic region.…”

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

“…A research article by Warren et al . () in The Journal of Physiology addresses the main question mark which looms over the pH hypothesis: how do HCs change cleft pH?…”

“…Warren et al . () outline a number of potential sources for extracellular protons: (i) extracellular carbonic anhydrase (CA) can create protons and bicarbonate from CO 2 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 .…”