Amiloride–Sensitive Conductance in Horizontal Cells That Could Regulate pH in the Synaptic Invagination of Photoreceptors

Abstract

Abstract: : Purpose: The messengers that mediate the generation of center–surround antagonistic receptive fields in the outer retina via inhibitory modulation at cone photoreceptor terminals remain unknown. Protons are potent Ca channel modulators at this synapse and cleft pH could be regulated by horizontal cells (HC). This work sought to identify a mechanism through which HC could modulate extracellular proton concentration. Methods: Feedback was measured as a change in presynaptic Ca concentration in retinal slices using Fluo–4 and recorded in isolated retina as the response of HCs to light. Isolated goldfish HC were recorded from with whole–cell patch techniques. Results: CNQX, which hyperpolarizes HC and reduces feedback inhibition, increased the depolarization–induced Ca signal of cone synaptic terminals by ∼60%. In contrast, CNQX reduced the Ca signal by 22 ± 9% (n = 10) when 50 µM amiloride was present. Amiloride did not change the Ca response to kainate. The CNQX–induced increase in Ca signal was also reduced by 0.25 mM Co (20 ± 10%; n = 9) and 0.25 mM Ni (13 ± 12%; n = 9), agents for which a selective inhibition of surround–induced feedback signals in the retina has been described. In goldfish retina, 200 µM amiloride decreased roll–back of the H1 cell light response from 12 ± 3% (n=4) to –10 ± 10% (n=4). The I–V relations of voltage–clamped HC showed inward rectification negative to –80 mV, an N–shape due to Ca channel activation positive to –20 mV, Na channel activation at –20 mV, and outward rectification positive to 0 mV. Over the physiological range (–70 to –20 mV), these cells had high input resistances (2.4 ± 1.2 GΩ; n = 6). Glutamate has complex effects on HC, which include modulation of inward and outward rectification. 100 µM glutamate increased conductance to 2.7 ± 0.9 nS, and amiloride reduced this to 1.7 ± 0.7 nS (n = 6), a 37% reduction.Conclusions:Feedback to cones is blocked by amiloride, possibly via an amiloride–sensitive conductance in HC that mediates cleft pH in a voltage–dependent manner. A proton conducting ion channel in the HC dendrites would provide a suitable pH regulating influence in the synaptic cleft since HC hyperpolarization would increase the inward driving force on protons, increasing cleft pH, and reducing proton–mediated inhibition of presynaptic Ca channels.

Keywords: retina: distal (photoreceptors, horizontal cells, bipolar cells) • electrophysiology: non-clinical • PH regulation/protons