Prolonged Photoresponses and Defective Adaptation in Rods of Gβ5^-/- Mice

Abstract

Timely deactivation of G-protein signaling is essential for the proper function of many cells, particularly neurons. Termination of the light response of retinal rods requires GTP hydrolysis by the G-protein transducin, which is catalyzed by a protein complex that includes regulator of G-protein signaling RGS9-1 and the G-protein beta subunit Gbeta5-L. Disruption of the Gbeta5 gene in mice (Gbeta5-/-) abolishes the expression of Gbeta5-L in the retina and also greatly reduces the expression level of RGS9-1. We examined transduction in dark- and light-adapted rods from wild-type and Gbeta5-/- mice. Responses of Gbeta5-/- rods were indistinguishable in all respects from those of RGS9-/- rods. Loss of Gbeta5-L (and RGS9-1) had no effect on the activation of the G-protein cascade, but profoundly slowed its deactivation and interfered with the speeding of incremental dim flashes during light adaptation. Both RGS9-/- and Gbeta5-/- responses were consistent with another factor weakly regulating GTP hydrolysis by transducin in a manner proportional to the inward current. Our results indicate that a complex containing RGS9-1-Gbeta5-L is essential for normal G-protein deactivation and rod function. In addition, our light adaptation studies support the notion than an additional weak GTPase-accelerating factor in rods is regulated by intracellular calcium and/or cGMP.

Gβ5 Is Required for Normal Light Responses and Morphology of Retinal ON-Bipolar Cells

“…1 D). The dominant recovery time constants ( D ) for control and G␤5 Ϫ/Ϫ rods were previously determined by suction recordings to be 260 and 2700 ms, respectively (Krispel et al, 2003). Using double flash ERG recordings, we found comparable D for the TG␤5L Ϫ/Ϫ (235 Ϯ 8 ms, n ϭ 15) and the control animals (189 Ϯ 12 ms, n ϭ 17).…”

“…In the RGS9 Ϫ/Ϫ mice, G␤5-L is undetectable, whereas G␤5-S level remains normal . Recordings from rods of G␤5 Ϫ/Ϫ (Krispel et al, 2003), RGS9 Ϫ/Ϫ , and R9AP Ϫ/Ϫ (RGS9 anchoring protein) (Keresztes et al, 2004) mice, which displayed similar and prolonged recovery of rod photoresponses, have established the role of G␤5-L/RGS9-1/R9AP as the GTPase accelerating protein complex (GAP) for transducin ␣-subunit (Gt␣). The GAP activity resides in the RGS domain of RGS9-1, whereas G␤5-L and R9AP confer intracellular stability, membrane affinity, and outer segment targeting modality to the complex Martemyanov et al, 2003;Keresztes et al, 2004).…”

Toward a unified model of vertebrate rod phototransduction

“…The time constant governing PDE*-inactivation [τ PDE in eqn. (A7c)] was increased by a factor of 10 Krispel et al, 2003;Martemyanov et al, 2003;Keresztes et al, 2004). (Burns et al, 2002).…”

Functional comparison of RGS9 splice isoforms in a living cell

“…Suction electrode recordings from dark-adapted rods were conducted as described in (25). The average response to a large number (Ͼ30) of flashes was considered to be in the linear range if its mean amplitude was less than 20% of the maximal response amplitude.…”

“…We next assessed the expression level of RGS9-2⅐G␤5 in transgenic rods. In this case, we concentrated on quantifying the long-splice isoform of G␤5, which exists exclusively as a constitutive equimolar complex with RGS9 in photoreceptors (6,9,21,25). We chose G␤5 rather than RGS9-2 to avoid the inherent differences in antibody recognition by individual RGS9 isoforms.…”