Investigations of Ligand-gated Ion Channels Using Caged Neurotransmitters Reveal Mechanism-based Approaches to Modulating Receptor Function

Abstract

Recognizing the problem of rapidly equilibrating membrane-bound proteins on cell surfaces with activating ligands due to the water layer that tightly adheres to the surface of spherical objects (1), we began to develop caged (2) neurotransmitters (3,4). A caged neurotransmitter can be equilibrated with receptors on a cell surface before photolysis releases the neurotransmitter in the microsecond time region (3,4). Using caged neurotransmitters in whole-cell current recordings enables the rate constants for the opening (kop) and closing (kcl) of the receptor channel, and thus the channel-opening equilibrium constant (Phe−1), to be measured without interference from diffusional delays or receptor desensitization.The rate constants and the channel-opening equilibrium constant of the excitatory nicotinic acetylcholine receptor (5) are altered in the presence of noncompetitive inhibitors, such as cocaine, as are those of a mutated inhibitory gamma-aminobutyric acid (GABA) receptor (6) linked to epilepsy (7). Based on the mechanisms of the two receptors, we developed compounds that bind with higher affinity to the open-channel than to the closed-channel conformation of each receptor, alleviating (8,9) the inhibition of the acetylcholine receptor and potentiating (10) the function of a mutated GABAA receptor linked to epilepsy.1. Landau & Lifshitz (1959) Fluid Mechanics (Pergamon, Oxford)2. Kaplan et al. (1978) Biochemistry 17, 19293. Milburn et al. (1989) Biochemistry 28, 514. Shembekar et al. (2007) Biochemistry 46, 54795. Hess et al. (2000) Proc. Natl. Acad. Sci. USA 97, 138956. Ramakrishnan. & Hess (2004) Biochemistry 43, 75347. Baulac et al. (2001) Nature Genetics 28, 468. Hess et al. (2003) Biochemistry 42, 61069. Chen et al. (2004) Biochemistry 43, 1014910. Krivoshein & Hess (2006) Biochemistry 45, 11632