Studying GCAP-independent Ca²⁺ Feedback in Mammalian Cones

Abstract

Purpose: Differences in GCAP- or Rec-mediated Ca²⁺-feedbacks are not sufficient to explain the differences between mouse rod and cone light adaptation. We sought to test the importance of GCAP- and Rec-independent Ca²⁺ -feedback in mammalian cones. This was done by mimicking light-induced drop in [Ca²⁺]_i in cones with reduced or absent cGMP synthesis modulation to prevent a high increase of cGMP in darkness.

Methods: Cone responses were recorded with transretinal ERG from mice in GNAT1^-/- background. Retinas were perfused with bicarbonate-buffered Locke’s solution that contained either 1.2 mM or low EGTA-buffered ~30 nM free [Ca²⁺] at 37 ^oC. To isolate the photoreceptor component of the ERG response 20 μM D, L-AP4 and 50 μM BaCl₂ were used. The change in the phototransduction gain caused by low Ca²⁺ exposure was defined as the factor by which the calculated single-photon response in low Ca²⁺ had to be scaled in order to match its leading edge with that in normal Ca²⁺.

Results: When control retinas were switched from normal to low Ca²⁺ the maximum cone response amplitude (r_sat) first increased several-fold and then declined together with significant deceleration and desensitization of flash responses producing 2-fold increase in dim flash response time-to-peak (t_p) and ~30-fold decrease in gain (n = 3). In GCAP^-/- cones t_p increased 15% and the gain decreased 10-fold when switched from normal to low Ca²⁺ (n = 4). Exposure of Rec^-/- cones to low Ca²⁺ caused several-fold transient increase in r_sat and 2.3-fold increase in t_p together with ~45-fold decrease in gain (n = 4). Rec^-/- cones in GCAP^+/- background showed 30% increase in t_p and 30-fold decrease in gain when they were exposed to low Ca²⁺ (n = 7). All mice except GCAP^-/- showed a prominent response recovery after light step onset and overshoot at light step offset in normal Ca²⁺. Both response features disappeared in low Ca²⁺.

Conclusions: Reducing expression level of GCAPs in mouse cones prevented excessive deceleration and desensitization of flash responses and enabled long stable recordings in low Ca²⁺. In addition, the degree of low Ca²⁺- induced decline in the gain of mouse cone phototransduction decreased with reduced expression of GCAPs. Thus, manipulation of [Ca²⁺]_o in GCAP^-/- background mice can be used to study GCAP-independent Ca²⁺ -feedback, such as CNG channel modulation, and Ca²⁺-independent feedback in mammalian cones.

Keywords: 648 photoreceptors • 439 calcium