Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor

Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B*) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B* is monophasic. The association rate coefficient of the monoliganded species AChR . B is k'(ass) = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B* from the monomer species AChR . B*. B (and AChR . B-2*), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R'(alpha) --> R ''(alpha) with the exchange dissociation constants: (k'(diss))(B) = 5.5(+/-1). 10(-5) s(-1) and (k ''(diss))B = 3(+/-1). 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B* by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B* are released from the AChR . B-2* species. The mechanism involves associative ternary intermediates (AChR . B*A, AChR . B*A(2) and AChR . B(2*)A(2)). The equilibrium constants (K-A) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R'(alpha) and R ''(alpha), respectively, are K'(A) = 1.1 . 10(-2) M and k'(-A) = 3 . 10(5) s(-1) and K ''(A) = 7.5 . 10(-2) M and k ''(-A) = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] less than or equal to 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] greater than or equal to 0.1 M.