Fig. 6.
The electrophysiological response of Slc26a7 knockout (KO) mouse retinal pigment epithelium (RPE) cells to 10 mM external thiocyanate (SCN−) is dramatically smaller compared with wild-type (WT) mouse RPE cells. A: families of whole cell currents evoked by voltage steps from a holding potential of −60 mV in an Slc26a7 KO mouse RPE cell in the absence and presence of 10 mM external SCN−. B: families of whole cell currents evoked by the same voltage-clamp protocol as in A in a WT mouse RPE cell in the absence and presence of 10 mM external SCN−. Note that the currents in WT mouse RPE exposed to SCN− are presented at a lower gain to account for their larger amplitude. C: summary of experiments in Slc26a7 KO mouse RPE cells similar to those in A showing current-voltage (I-V) plots of instantaneous current obtained in the absence (closed circles) and presence (open circles) of 10 mM external SCN−. Symbols represent means, and bidirectional error bars represent SE (n = 19 cells from 5 mice of both sexes, 7–12 wk old). Ten millimolar SCN− produced significant increases in current at voltages in the ranges of −114.5 to −84.5 mV and −4.5 to +55.5 mV (P < 0.02; Šidák’s multiple comparisons test). D: summary of experiments in WT mouse RPE cells similar to those in B showing I-V plots of instantaneous current obtained in the absence (closed circles) and presence (open circles) of 10 mM external SCN−. Symbols represent means, and bidirectional error bars represent SE (n = 9 cells from 1 male and 1 female mouse, 18.6 wk old). Ten millimolar SCN− produced significant increases (P < 0.002; Šidák’s multiple comparisons test) in current at voltages in the ranges of −114.5 to −84.5 mV and −24.5 to +55.5 mV. E: comparison of normalized outward conductance of RPE cells isolated from Slc26a7 KO mice (left, n = 19 cells from 5 mice of both sexes, 7–12 wk old) and WT mice (right, n = 13 cells from 6 mice of both sexes, 6.4–24 wk old) and in the absence (open symbols) and presence (gray symbols) of 10 mM external SCN−. Horizontal lines and error bars represent means ± SD. The difference in conductance between Slc26a7 KO mouse RPE cells and WT mouse RPE cells was significant in the absence (P < 0.05) and presence of external SCN− (P < 0.00001, 2-tailed unpaired t tests). Data for WT mouse RPE cells include the results from 8 cells from our previous study on Slc26a7+/+ mice (10). F: comparison of the relative permeability for SCN− of RPE cells isolated from Slc26a7 KO mice (left) and WT mice (right) determined at holding potentials (HPs) of −60 mV (open symbols) and −120 mV (gray symbols). Permeability ratios (PSCN/PCl) were calculated from the change in reversal potential (ΔErev) of instantaneous currents produced by replacing 10 mM external Cl− with SCN− using the Goldman–Hodgkin–Katz equation (Eq. 3). When HP = −60 mV, PSCN/PCl values in Slc26a7 KO mouse RPE cells [30.1 ± 3.7 (SE), n = 10 cells from 3 mice of both sexes, 7–12.5 wk old] were significantly lower than in WT mouse RPE cells [75.4 ± 8.3 (SE), n = 13 cells mice of both sexes, 6.4–18.6 wk old; P < 0.0002, 2-tailed unpaired t test]. Hyperpolarization of HP to −120 mV did not change the PSCN/PCl value in Slc26a7 KO mouse RPE cells significantly [34.2 ± 5.3 (SE), n = 10 cells from 3 mice of both sexes, 7–12.5 wk old, P > 0.05 compared with values obtained at HP = −60 mV, 2-tailed paired t test] but, as previously shown (10), resulted in a dramatically higher PSCN/PCl value in WT mouse RPE cells [437.4 ± 41.6 (SE), n = 13 cells mice of both sexes, 6.4–18.6 wk old; P < 0.0002, 2-tailed paired t test]. Data for WT mouse RPE cells include the results from 4 cells from our previous study on Slc26a7+/+ mice (10). The apparent PSCN/PCl value is underestimated in WT mouse RPE cells held at −60 mV because of the depolarizing effect of intracellular SCN− accumulation on Erev, an effect that is minimized at HP = −120 mV (10). The insensitivity of the PSCN/PCl value on HP in Slc26a7 KO RPE cells suggests that in the absence of SLC26A7 the intracellular accumulation of SCN− is relatively minor at negative potentials.