Light response of retinal ON bipolar cells requires a specific splice variant of Galpha(o)

Abstract

Glutamate released onto retinal ON bipolar neurons binds to a metabotropic receptor to activate a heterotrimeric G-protein (G(o)) that ultimately closes a nonspecific cation channel. Signaling requires the alpha subunit (Galpha(o)), but its effector is unknown. Because Galpha(o) is transcribed into two splice variants (alpha(o1) and alpha(o2)) that differ in the key GTPase domain, the next step in elucidating this pathway was to determine which splice variant carries the signal. Here we show by reverse transcription-PCR and Western blots that retina expresses both splice variants. Furthermore, in situ hybridization and immunostaining on mouse retina deficient in one splice variant or the other show that both alpha(o1) and alpha(o2) are expressed by ON bipolar cells but that alpha(o1) is much more abundant. Finally, electroretinography performed on mice deficient for one splice variant or the other shows that the positive b-wave (response of ON bipolar cells to rod and cone input) requires alpha(o1) but not alpha(o2). Thus, the light response of the ON bipolar cell is probably carried by its strongly expressed splice variant, Galpha(o1).

Fig. 1.

Retina expresses both splice variants of α_o. A, Primers for amplifying α_o1 and α_o2 mRNA; restriction enzymes cutting sites are also indicated. B, mRNA from rat retina was reverse transcribed and amplified using the specific primers shown in A. In lanes 2 and4, the PCR products were cut with restriction enzymes.C, Dot blots show that α_o1 antibody does not recognize the α_o2 peptide (α_o2pep) and that α_o2 antibody does not recognize the α_o1peptide (α_o1 pep). Neither antibody reacted with bovine serum albumin (BSA). D, Western blots of whole rat retina show, for α_o1, a single prominent band at ∼43 kDa and, for α_o2, a prominent band at ∼40 kDa plus a weak band at ∼50 kDa.

Fig. 2.

Bipolar cells strongly stain for the α_o1 splice variant. A, B,In situ hybridization (rat). Antisense probe gave labeling in the INL and in the ganglion cell layer (GCL). Strongest labeling is in the upper tier of the INL, the location of rod bipolar somas. Sense probe gave low background. Left panels, Differential interference contrast shows the retinal layers of the section in A.PR, Photoreceptors. C–E, Immunostaining with the α_o1-specific antibody visualized with 3,3′-diaminobenzidine (rat). C, Staining is strong in bipolar dendrites (Den) in OPL and weaker in bipolar somas (BP) in the INL and in the IPL. D, Staining with the preimmune serum was negative. E, Staining with the antibody preabsorbed with the α_o1peptide was also negative. F–H, Immunostaining with the α_o1-specific antibody visualized with FITC (mouse).F, In the OPL and INL, staining in wild-type mouse resembles that in rat. In the IPL, note the thin bands of higher intensity (black arrows), a thicker band of lower intensity (brackets), and a band devoid of staining (white arrow). G, H, No staining above general background was observed when the same antibody was applied, respectively, to an α_o1 or α_o1+2 null mouse. ko, Knock-out;wt, wild type.

Fig. 3.

All ON cone bipolar cells stain for α_o1 (rat). Left, All rod bipolar somas (stained for PKC) also stained for α_o1 (double arrowhead), but some somas stained only for α_o1(long arrow). In the OPL (bracketed), rod bipolar dendrites stained for both PKC and α_o1(arrowhead), but some dendrites stained only for α_o1 (short arrow). These probably belong to ON cone bipolar cells. Right, All ON bipolar somas were identified by staining with the monoclonal antibody for α_o. All of these somas also stained for α_o1. Also, in the IPL, the two staining patterns were the same, indicating that the retina expresses primarily the α_o1 splice variant.

Fig. 4.

Bipolar cells and stratum 1 of the IPL weakly express the α_o2 splice variant. A,B, In situ hybridization (rat). Antisense probes specific for α_o2 message hybridized strongly in the INL and weakly in the ganglion cell layer (GCL). Sense probes gave weak, diffuse background. Left, Differential interference contrast for retinal section inA. C–F, Immunostaining of mouse retina, visualized with FITC. D–F were captured using the same confocal laser intensity and gain parameters. C, Antibody against the CT peptide of α_o1 applied to the α_o1 null retina faintly stained the OPL, bipolar somas (BP), and stratum 1 of the IPL (brackets). D, Antibody against the NT peptide applied to the α_o1 null retina gave similar staining: faint in the OPL, bipolar somas, and puncta throughout the IPL, plus more intense staining in stratum 1. E, NT antibody applied to α_o knock-out (KO) retina gave weak diffuse background staining. F, NT antibody applied to α_o2 knock-out retina gave strong labeling, resembling that obtained with this and other anti-α_o antibodies in wild-type retina.

Fig. 5.

Rod bipolar and some cone bipolar cells stain for Gα_o2. Rod bipolar cells were identified by staining for PKC; staining for α_o2 was achieved by staining an α_o1 null mouse with anti-Gα_o-CT. All bipolar somas stained for PKC also stained for α_o2(double arrowhead), but some somas were stained only for α_o2 (arrow).

Fig. 6.

Rod- and cone-driven b-waves are present in the electroretinogram of Gα_o2 null mouse but absent from the Gα_o1 null mouse. A, Animals dark adapted for 2 hr were stimulated with dim flashes that elicited a rod-driven, corneal-positive b-wave in the wild-type mouse and in Gα_o2 null but not in the Gα_o1 null. Flash intensities estimated as photoisomerizations (R*) per rod (Φ), and number of responses (n) averaged for each trace were as follows: Φ = 9; n = 18. B, Dark-adapted animals were stimulated with an intense flash isomerizing ∼0.1% of the rhodopsin. This elicited in wild-type and Gα_o2 null mice a negative a-wave (shading), followed by a positive b-wave. The Gα_o1 null mice showed an a-wave (although somewhat reduced) but no b-wave. Φ = 10⁵;n = 4. C, Mice were adapted to a background (white light, 9100 R* rod⁻¹ sec⁻¹) that completely suppressed the rod cGMP-activated current. Rods were then stimulated with an intense white flash that isomerized ∼0.4% of the M-cone pigment and 0.03% of the UV-cone pigment. All of the animals showed a cone-driven a-wave. A typical cone-driven b-wave (positive-going response with superimposed oscillations, peaking ∼70–90 msec after the flash) was present in wild-type and α_o2 null mice but was absent in the Gα_o1 null animals. For all of the records, n = 16. KO, Knock-out;WT, wild type.