Molecular kinetics of metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) belong to class C G-protein-coupled receptors (GPCRs). They are characterized by a large extracellular domain which is composed of the ligand-binding domain (LBD), followed by a cysteine rich-domain (CRD) that ends up at the transmembrane domains (TDM). After glutamate binding, each subunit of the mGluR dimer move towards one another (intermolecular rearrangement) and then intramolecular rearrangements follow. mGluRs are organized either as homodimers or heterodimers. Eight members belonging to mGluRs are classified in three groups: group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3), and group III (mGluR4, mGluR6, mGluR7 and mGluR8). Activation kinetics of ligand-activated GPCRs has been reported to be in the range of 10-80 ms, with mGluR1 being the fastest receptor measured so far, however, it was limited by the method that was used in the respective studies. On the other hand, activation kinetics of rhodopsin, a light-activated GPCR, is in the range of 1 ms, measured with optical methods, not applicable to other GPCRs. Therefore, we used the a confocal patch-clamp fluorometry (cPCF) combined with fast concentration jumps and a high-resolution laser scanning microscope (LSM) to analyze the real activation kinetics of mGluR1 in outside-out membrane patches expressing mGluR FRET sensors. Our data show fast intermolecular activation kinetics of 1 ms, about ten times faster than data published previously and our value is not limited by our technique. The kinetics within a receptor subunit (A-sensor) was around 20 times slower for both activation and deactivation kinetics. The observed deactivation kinetics was determined to be in the time range of tens of ms for the E-sensor, and hundreds of ms for the A-sensor. Furthermore, we used same approach to create FRET sensors for all members of mGluRs, mGluR1 to mGluR8 and systematically analyzed dimerization and kinetics for all 36 homo- and heterodimeric forms.