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Z. Wang, X. Wen, C. Makino, J. Lem; Functional Analysis of Palmitoylation-defective Rhodopsin in Knock-in Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4512.
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Purpose: To investigate the physiologic role of rhodopsin palmitoylation in vivo, we generated a palmitoylation-defective rhodopsin knock-in (Palm-/-) mouse and then assessed the phototransductive capacity of their retinal rods. Methods: The palmitoylated cysteines at positions 322 and 323 of rhodopsin were mutated to non-palmitoylatable threonine and serine residues, respectively. To prevent complications arising from a change in the level of expressed rhodopsin protein, the mutations were targeted to the endogenous rhodopsin gene. Retinal morphology and rhodopsin localization were evaluated with light microscopy. Protein levels were determined by Western analysis or spectrophotometrically. Transducin activation was tested in rod outer segment preparations by measuring the extent of GTPγS incorporation following an exposure to light. Physiological function was analyzed using the single cell suction electrode recording method on Palm-/- and wild type (WT) mice at 5 to 7 weeks of age. Results: Gross retinal morphology in one- and three- month-old Palm-/- animals was indistinguishable from that of age-matched WT controls, except that the outer segments of mutant rods were slightly shorter. Immunohistochemical examination confirmed that the mutant rhodopsin localized appropriately to the outer segment layer in Palm-/- retinas. No differences in the levels of rhodopsin, rhodopsin kinase, arrestin or transducin α-subunit were detected in WT and Palm-/- mice. Biochemical assays indicated a reduction in transducin activation in Palm-/- mice compared to WT controls. Single-cell recordings revealed normal dim flash response kinetics in Palm-/- rods, however, flash sensitivity was reduced two-fold. This reduced sensitivity can not be explained by diminished photon capture due to the slight shortening of outer segment length. Conclusions: Rhodopsin palmitoylation does not appear to alter rhodopsin expression nor does it affect the intracellular transport of the rhodopsin protein. Biochemical and electrophysiological results suggest that rhodopsin lacking palmitoylation is capable of supporting phototransduction but with reduced signaling efficiency. Consequently, Palm-/- rods are less sensitive than normal.
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