Structural characterization of agonist and antagonist-bound acetylcholine-binding protein from Aplysia californica.

Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition.

The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzymatic kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K+ over Rb+ and to its blockage by Cs+ and by Ca2+. The channel was investigated by patch-clamp techniques over a range of membrane voltages (Vm, referred to an extracytoplasmic electrical potential of zero) from -200 mV to +200 mV under various ionic conditions (0 to 300 mM K+, Rb+, Cs+, Ca2+, and Cl-) on the two sides of isolated patches. The experimental data are apparent steady-state current-voltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs+. The used model for K+ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K(+)-loaded and charged, facing the cytoplasm, (2) K(+)-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. Vm enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10(6)s-1 for zero voltage and 1 M substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 15,800. For the additional presence of an alternate transportee (here Rb+), the model can be extended in an analog way by another two states ((5) Rb(+)-loaded and charged, facing cytoplasm, and (6) Rb(+)-loaded and charged, facing vacuole) and six more rate constants (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs+ and Ca2+). k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 M-1 x exp(Vm/(64 mV)), kvac(Ca2+): 10 M-1 x exp(-Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 M-2 x exp(-Vm/(12.25 mV)).(ABSTRACT TRUNCATED AT 400 WORDS)