Fig 1.
CaV channels in cone photoreceptors of mice, rats, and guinea pigs are maintained in a pH-mediated, tonically inhibited state by GABAR activation.
A. Image of a mouse cone filled with Lucifer yellow via the patch clamp electrode. B–D. GABAR antagonist picrotoxin (100 μM) increases peak calcium current amplitude in mouse, rat, and guinea pig cones, maintained in mesopic conditions. Fine dashed lines indicate zero current. E,F. Sample current traces and I–V relations of a rat cone before, during, and after application of picrotoxin. G. The CaV channel activation curve for the cell in (E) reveals a leftward shift in the V½ in picrotoxin (V½ = −41 mV) versus control (V½ = −37 mV). H–J. Same experiment in E–G in the same cell while clamping pH to 7.4 with the pH buffer Hepes (10 mM), eliminating the effect of picrotoxin on CaV channel activation (V½ = −39 mV). This implies a tonic GABA inhibition that is pH dependent. Underlying data of cells in this figure can be found in S1 Data. CaV channel voltage-gated Ca2+ channel; I–V, current–voltage; V½, half-maximal activation voltage.
Fig 2.
GABARs containing ρ-subunits mediate the modulation of cone CaV channel currents, and ρ-subunits are expressed in horizontal cell synaptic tips.
Effects of GABAR blockers on CaV channel currents in cones in guinea pig retinal slices. A–C. The ρ-subunit–containing GABAR antagonist TPMPA (50 μM) shifts CaV channel activation in guinea pig cones to more negative voltages (V½ = −29 versus V½ = −39 for cell shown). D–F. The GABAAR antagonist gabazine (10 μM) has no effect on CaV channel activation. G. Graphic to show all recordings in the figure are made from cones. H. Summary showing the effects of TPMPA (n = 5), gabazine (n = 7), and the GlyR antagonist strychnine (100 μM; n = 6) on CaV channel activation. I. GABAR ρ2 subunit (blue) and calbindin (red; J) immunoreactivity in mouse retina with maximum intensity projections. The ρ2 subunit is strongly expressed in the tips of the horizontal cell processes (merged in K and enlarged insets in I: cone pedicle, upper left; rod spherules, lower right), where they enter the photoreceptor terminals. Underlying data of cells in this figure can be found in S1 Data. CaV channel, voltage-gated Ca2+ channel; GABAR, GABA receptor; GlyR, glycine receptor; TPMPA, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid.
Fig 3.
Tonic horizontal cell release of GABA activates ρ-subunit containing GABARs on its own cellular membrane, which indirectly inhibits cone CaV channels.
A. Patch clamp recording of a tdTomato labeled mouse horizontal cell in a slice. B. Currents elicited in a horizontal cell bathed in 50 μM CNQX and 10 mM Hepes by voltage steps in control (top) and in 50 μM TPMPA (below). C. Average I–V relation of TPMPA-subtracted current for five cells shows a linear component reversing near −67 mV (dotted line is linear fit excluding values at −39 and −29 mV). Width of orange line shows the standard deviation for the 5 cells. D–F. Same experiment as in A-C but in Cx57-VGAT-KO mouse horizontal cells [37]. Mouse horizontal cells lacking VGAT are unaffected by TPMPA. G. Patch clamp recording of cone in wild-type mouse. H. Current in the absence and presence of TPMPA. I–V relations (I) and activation curves (J) of the cell in (H) shows that the activation midpoint shifted from −39 mV to −44 mV with TPMPA application. K–N. Same experiment in A–C in a cone of a Cx57-VGAT-KO mouse. Unlike cones in wild-type retinas, the activation midpoint is unaffected by TPMPA in the VGAT-deleted cones. O–P. VGAT (blue) and calbindin (red) immunoreactivity in wild-type (O) and Cx57-VGAT-KO (P) mouse retinas (scale bar = 10 μm). Sparse labeling for VGAT in (P) represents projections from cells in the inner retina. The super-resolution confocal image (maximum intensity projection) in O shows VGAT immunolabeling in horizontal cell endings that correspond to the same cellular compartment the GABAR ρ2 subunits were seen localized in Fig 2I–2K. Underlying data of cells in this figure can be found in S1 Data. CaV channel, voltage-gated Ca2+ channel; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; GABAR, GABA receptor; I–V, current–voltage; KO, knockout; TPMPA, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid; VGAT, vesicular GABA transporter.
Fig 4.
Activation of exogenously expressed Cl−/HCO3− channels on horizontal cell membranes is sufficient to inhibit CaV channels in mouse cones.
A. AAV-7m8-PSAM-GlyR-IRES-GFP construct. Cre-expressing horizontal cells transduced with this virus express Cl−/HCO3− permeable GlyR complexes that can be activated by the orthogonal ligand PSEM. B. GFP fluorescent somata (reporter for AAV-7m8-PSAM-GlyR). C. Calbindin immunostaining of horizontal cell bodies in a whole-mount retina. D. Merged image focused on the distal INL. Most calbindin-labeled cell bodies express GFP, and nonspecific staining of blood vessels is visible. E. Patch clamp recording of a cone of a Cx57-PSAM-GlyR mouse. F. Currents elicited by the voltage steps shown in the absence (top) and presence (bottom) of 200 nM PSEM. I–V plots (G) and activation curves (H) of the cell in (F) reveal a rightward shift of activation midpoint with PSEM application, in a manner similar to the GABA agonist muscimol in the following figure. Underlying data of cells in this figure can be found in S1 Data. AAV, adeno-associated virus; GFP, green fluorescent protein; GlyR, glycine receptor; INL, inner nuclear layer; IRES, internal ribosome entry site; I–V, current–voltage; PSAM, pharmacologically selective actuator module; PSEM, pharmacologically selective effector molecule.
Fig 5.
GABAR inhibition of cone CaV channels depends on horizontal cell [Cl−]i.
A. Gramicidin-perforated patch clamp recordings of tdTomato-labeled mouse horizontal cells. B. Currents elicited by the voltage steps shown in control (top) and in the presence of 100 μM muscimol. C. Average I–V relations of muscimol-subtracted current (n = 5) shows the principle linear component reversing at −28 mV (dotted line is linear fit of mean subtracted currents). D. Whole-cell patch clamp recording of a mouse horizontal cell with a K+-gluconate-based internal solution containing 41 mM chloride. E. Time course of membrane potential, recorded under current-clamp, during response to muscimol (approximately 1 min application). F. Patch clamp recording of a mouse cone. G. Currents elicited in mouse cones by the voltage steps shown in the absence (top) and presence (bottom) of muscimol. H. I–V relations show smaller calcium currents in the presence of muscimol. I. Activation curves of the cell in (G) reveal a rightward V½ shift in muscimol. J–M. Same experimental paradigm as in F–I in mouse cones bathed in the NKCC blocker bumetanide. K. Currents in the absence and presence of muscimol with bumetanide pretreatment (30 min). I–V curves (L) and activation curves (M) of the cell in (K) reveal a slight leftward V½ shift with muscimol in bumetanide-treated retinas. Underlying data of cells in this figure can be found in S1 Data. CaV channel; voltage-gated Ca2+ channel; GABAR, GABA receptor; I–V, current–voltage; NKCC, Na+/K+/Cl- cotransporter; V½, half-maximal activation voltage.
Fig 6.
Blocking Na+/H+ exchangers with the selective inhibitor cariporide disinhibits cone CaV channels and eliminates the disinhibitory effect of TPMPA.
A. Patch clamp recording of a mouse cone. B. Currents elicited by voltage steps shown in a mouse cone bathed in control bath alone (top), after adding 10 μM cariporide (middle), and after adding 50 μM TPMPA with the cariporide (bottom). C. I–V relations show larger CaV channel currents in the presence of cariporide, similar to the effects of the GABACR antagonist TPMPA (cf. Fig 3G–3J). D. The cone CaV channel activation curve shifts to a more negative potential during cariporide application, from −15.8 mV to −21.9 mV. E–G. I–V relations show little effect of TPMPA on CaV channel current in the same mouse cone now bathed continuously with cariporide. H. In the presence of cariporide, TPMPA fails to shift CaV channel activation curve to more negative potentials (−21.9 mV to −21.9 mV). Underlying data of cells in this figure can be found in S1 Data. CaV channel, voltage-gated Ca2+ channel; GABAR, GABA receptor; I–V, current–voltage; TPMPA, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid.
Fig 7.
Polarity of cone CaV channel modulation depends on horizontal cell membrane potential.
A. Currents elicited by the voltage steps shown in the absence (top) and presence (bottom) of 100 μM muscimol during whole-cell patch clamp recordings of a cone in a guinea pig retinal slice in low mesopic conditions (G). B. Current voltage relations show smaller calcium currents in the presence of muscimol. C. Activation curves of the cell in (A) reveals V½ shifts from −36 to −29 mV with muscimol application (cf. Fig 4F–4I). D–F. Same experiment in A–C in the presence of CNQX (50 μM). Under these conditions, muscimol shifts V½ from −21 to −34 mV. G. Graphic to show that the recordings are made from cones. H. A summary plot of ΔV½ elicited by muscimol application in control (n = 6) and in CNQX (n = 4) demonstrates that the polarity of muscimol’s effect is dependent on glutamatergic depolarization of horizontal cells. Underlying data of cells in this figure can be found in S1 Data. CaV channel, voltage-gated Ca2+ channel; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; V½, half-maximal activation voltage.
Fig 8.
Modulation of cone presynaptic CaV channels by horizontal cell regulation of cleft pH.
A. Acidification of the cleft during horizontal cell depolarization (“Vm+”) in relative dark. HCO3− efflux via GABARs decreases due to reduced driving force (EHCO3− close to Vm+). Continuous H+ extrusion occurs to offset intracellular acidification caused by metabolic activity. Inhibited Ca2+ influx into cone reduces glutamate release. B. Alkalization of the synaptic cleft occurs during strong hyperpolarization (“Vm‒”) of the horizontal cells due to increased driving force on HCO3− efflux via GABARs and reduced H+ efflux, disinhibiting cone CaV channels and increasing glutamate release. CaV channel, voltage-gated Ca2+ channel; GABAR, GABA receptor; Vm, membrane potential.