NMR measurement of the off rate from the first calcium-binding site of the synaptotagmin IC2A domain

The off rate from the first calcium-binding site of the C(2)A domain of synaptotagmin 1, a putative calcium receptor in neurotransmitter release, has been determined by N-15-nuclear magnetic resonance relaxation dispersion measurements. The exchange rate was obtained by fitting the dependence of the transverse relaxation rates on the interval between 180degrees pulses in relaxation-compensated CPMG experiments at 3.2 muM calcium concentration. The measured k(ex) is 2.0 X 10(3) s(-1). The calcium on rate of 3.5 +/- 1 X 10(7) s(-1), determined from the measured off rate and the dissociation constant (5.3 X 10(-5) M), is close to the diffusion limit. These results are consistent with the proposed role of synaptotagmin I as a calcium sensor in release, but suggest that additional factors may help to accelerate the diffusion of Ca2+ to the sensor. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.