Fig. 7.
LPHN3ΔE5 variant reduces cone synaptic release and inhibits calcium channel function. (A) Schematic of retinal slice recording from cone photoreceptors. The inset shows a cone filled with sulfarhodamine B in WT retinal slice. (B) Representative traces documenting paired-pulse inhibition of ICa in a control WT cone (Top). Application of a depolarizing test step (25 ms, −70 to −10 mV) evoked a transient inward current followed by a brief outward current deflection reflecting inhibition by vesicular protons. Due to the depletion of releasable vesicles by the first pulse, this proton-mediated inhibition was abolished during the second pulse applied 50 ms later. (Bottom) Representative traces documented showing limited paired-pulse inhibition of ICa in a ΔE5 cone. (C) Quantification of the paired-pulse ratio (amplitude of ICa evoked by the first pulse/amplitude of ICa evoked by the second pulse) between control WT (n = 4) and ΔE5 (n = 7) cones (**P < 0.01, unpaired Student’s t test, and error bars are SEM values). The larger paired-pulse ratio in ΔE5 cones indicates that less glutamate was released in these cones. (D) Representative traces of ICa from ΔE5 (red trace) and control WT cones (black trace) evoked by a ramp voltage protocol (−102.5 to +47.5 mV, 0.5 mV/ms). (E) ICa peak amplitude measured with ramp voltage protocols was reduced significantly in ΔE5 cones (red, n = 8; *P < 0.05, unpaired Student’s t test, and error bars are SEM values) relative to control WT cones (n = 4). (F) Analysis of the midpoint of activation voltage of calcium channels in cones of WT (n = 4) and ΔE5 (n = 7) retinas. Data are shown as mean ± SEM, unpaired Student’s t test. (G) Representative images of WT and ΔE5 retina sections immunostained with antibodies indicated (Scale bar, 5 μm). (H) Quantification of the mean fluorescence intensity of CaV1.4 at cone synapse of WT (black, n = 3) and ΔE5 (red, n = 3) retinas. Error bars are SEM values and unpaired Student’s t test. (I) Quantification of the area occupied by CaV1.4 at cone synapse of WT (black, n = 3) and ΔE5 (red, n = 3) retinas. Error bars are SEM values and unpaired Student’s t test. (J) Analysis on the ultrastructure of cone synapse of WT and ΔE5 retina by EM. Cone terminals are labeled in pale green, HC processes in blue, and bipolar dendrites in red (Scale bar, 1 μm).