Abstract
Purpose:
Kir7.1, an inwardly rectifying K+ channel, is expressed in several ion-transporting epithelia including the retinal pigment epithelium (RPE). Mutations in the KCNJ13 gene that encodes for Kir7.1 cause Snowflake Vitreoretinal Degeneration (SVD) and Leber Congenital Amaurosis (LCA). These allelic vision disorders are characterized by an abnormal ERG. The RPE Kir7.1 primarily helps K+ recycle through the apical membrane to maintain subretinal space ionic homeostasis. In this study we sought to elucidate whether inhibition of Kir7.1 impacts the mouse scotopic ERG.
Methods:
Standard ERGs were performed on dark-adapted 4 wk old C57/BL6 mice from an intensity of 0.03 to 30 log cd-s/m2 using the HMsERG system (Ocuscience, Kansas, USA) under Ketamine, Xylazine, and acepromazine (90:7.5:1.75) mg/kg anesthesia IP. Kir7.1 inhibitors (Lentiviral particles of shRNA, control shRNA, VU590, and VU608) were delivered via intravitreal injections. Amplitudes for the a-, b-, and c-waves were measured and averaged. Isolated retina ERG was recorded under constant perfusion of AMES medium supplemented with test compounds using the ex vivo ERG system (Ocuscience). We performed standard whole-cell patch-clamp on single mouse RPE cells to compare Kir current measurements. The student t-test was used for statistical comparison and a p-value < 0.05 were deemed to be significant.
Results:
Knock-down of Kir 7.1 expression with shRNA reduced the a-, b- and c-wave amplitudes of the mouse ERG. The c-wave was reduced by 49 ± 0.2 % (p<0.001) compared to control shRNA (c-wave 4 ± 0.3 % p=0.69). Kir7.1 channel blocker VU590 (50uM), but not the inactive analog VU608 (50uM), selectively attenuated the ERG amplitudes. In ex vivo recordings, however, VU590 had no effect on the isolated retina ERG a- and b-wave amplitudes. Whole-cell inward current measured at -160 mV in isolated RPE cells was blocked by VU590 (35 ± 3%, p<0.005) but not VU608 (5 ± 2%, p=0.85).
Conclusions:
We have shown that Kir7.1 knockdown by shRNA or inhibition by VU590 affects the mouse scotopic ERG. This finding correlates with the ERG phenotype seen in SVD and LCA16 patients when there is a loss of Kir7.1 function. We suggest that the ERG abnormalities found in these disorders result at least in part from a failure of communication between the RPE and photoreceptors.