Altered agonist sensitivity and desensitization of neuronal mGluR1 responses in knock-in mice by a single amino acid substitution at the PKC phosphorylation site

mGluR1 and mGluR5 of the metabotropic glutamate receptor family are coupled to inositol trisphosphate-Ca²⁺ signal cascades and evoke distinct Ca²⁺ responses in neural cells and heterologously expressing cells. In heterologous cells, stimulation of recombinant mGluR1 evokes a single-peaked Ca²⁺ response whereas mGluR5 elicits an oscillatory Ca²⁺ response. The distinct Ca²⁺ responses are interchangeable by single amino substitution of aspartate for threonine at the corresponding position of the carboxy-terminal cytoplasmic regions of mGluR1 and mGluR5, respectively. In this investigation, we generated knock-in mice, termed mGluR1 D854T mice, in which aspartate of mGluR1 was replaced with threonine. We examined the effect of this D854T substitution on Ca²⁺ and current responses mediated by mGluR1 in cultured cerebellar Purkinje cells. Stimulation of mGluR1 D854T by a group 1 mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG) evoked, as in wild-type mGluR1, only single-peaked Ca²⁺ responses as measured by Ca ²⁺ fluorometric analysis. We then examined mGluR1-induced inward currents carried by nonselective cation channels during whole-cell recordings from cultured Purkinje cells. The mGluR1 D854T mutation abolished the responsiveness of mGluR1 to low concentrations of DHPG (0.5-500 nM) and reduced its desensitization during prolonged agonist application. mGluR1 D854T homozygous mutants showed no apparent behavioural abnormality as analysed by motor movement tests. These results indicate that, although additional modulatory mechanisms seem to be required to produce oscillatory Ca²⁺ responses of mGluR1, the single amino acid substitution at position 854 of mGluR1 is capable of influencing the kinetics of neuronal mGluR1 responses, most probably through PKC-mediated phosphorylation.