Construction of Mutant Achr Subunits and Expression of Receptors

Supplementary Methods

Construction of mutant AChR subunits and expression of receptors The cDNAs encoding human -, -, -, and -subunits, subcloned into the CMV-based mammalian expression vector pRBG4, were described previously1. Mutations were made using the QuickChange Site- Directed Mutagenesis kit (Stratagene) and confirmed by sequencing the entire coding region. BOSC 23 cells2 were transfected with either wild-type or mutant receptor subunit cDNAs by calcium phosphate precipitation as described previously3. A plasmid encoding green fluorescent protein (pGreen lantern) was co-transfected with receptor plasmids to identify transfected cells under fluorescence optics. Patch-clamp recordings To record single channel currents, cells 1-2 days following transfection were rinsed and maintained in a bath solution containing (in mM): KCl 142, NaCl 5.4, CaCl2 1.8, MgCl2 1.7, and HEPES 10 (pH was adjusted to 7.4). Pipettes were filled with the same solution containing specified concentrations of ACh. Single channel currents were recorded in the cell-attached patch configuration using an Axopatch 200A patch clamp amplifier (Axon Instruments, Inc.). Currents were low-pass filtered at 50 kHz, digitized at 200 kHz (ITC-16, Instrutech Corp.), recorded to hard disk using the program Acquire (Bruxton Corp.), and unitary events were detected using the half-amplitude threshold criterion at a final bandwidth of 10 kHz. [125I]-bungarotoxin binding Expression of wild-type and mutant receptors on the cell surface was determined by measuring [125I]- bungarotoxin (Perkin Elmer® Life and Analytical Sciences) binding to intact cells as described previously4. For all mutants the ability of ACh to bind to the receptor was verified by competition against the initial rate of [125I]-bungarotoxin binding.

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