Potentiation of exocytosis by phospholipase C-coupled G-protein-coupled receptors requires the priming protein Munc13-1.

The vesicle priming protein Munc13-1 is regulated by diacylglycerol (DAG) and is therefore hypothesized to play a role in the control of neurotransmitter release by phospholipase C (PLC)-coupled receptors. We combined voltage-clamp recordings of voltage-gated Ca2+ channels (VGCCs) and high-resolution capacitance measurements to investigate the mechanism of receptor-mediated modulation of exocytosis in bovine chromaffin cells. Activation of endogenous H1 G(q)-protein-coupled receptors (G(q)PCRs) by histamine potentiated stimulus-coupled secretion despite concurrently inhibiting Ca2+ influx through VGCCs. Histamine increased the size of the readily releasable pool of vesicles and in particular a subpool of fusion-competent vesicles localized in close proximity to VGCCs. Pharmacological characterization showed that potentiation of exocytosis depended on the activation of PLC but not protein kinase C. Overexpression of wild-type Munc13-1 by adenoviral infection had no effect on histamine-induced potentiation per se, whereas DAG-insensitive Munc13-1(H567K) completely abolished it. This is the first endogenous mammalian G(q)PCR signaling pathway identified that engages Munc13-1 to increase stimulus-coupled secretion by recruiting vesicles to the immediately releasable pool. G(q)PCRs are therefore able to control exocytosis at the level of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation to produce rapid, short-term potentiation of the secretory output of neurons and endocrine cells.

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins.

Green fluorescent protein and its variants are frequently used as Förster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.

Engineering Dictyostelium discoideum myosin II for the introduction of site-specific fluorescence probes.

Dictyostelium discoideum is a useful host for the production of constructs for the analysis of structure-function relationships of myosin. Here we describe the use of myosin II constructs containing a single tryptophan residue, at different locations, for probing events at the nucleotide binding site, the relay loop and the communication path between them. GFP fusions have also been expressed at the N- and C-termini of the myosin motor to provide sensitive probes of the actomyosin dissociation reaction in microscope-based kinetic assays. We report on the fluorescence anisotropy of these constructs in the context of their use as resonance energy transfer probes.