Abstract

Human α4β2 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus laevis oocytes or transfected cell lines are present as a mixture of two stoichiometries, (α4)₂(β2)₃ and (α4)₃(β2)₂, which differ depending on whether a β2 or α4 subunit occupies the accessory subunit position corresponding to β1 subunits of muscle AChRs. Pure populations of each stoichiometry can be expressed in oocytes by combining a linked pair of α4 and β2 with free β2 to produce the (α4)₂(β2)₃ stoichiometry or with free α4 to produce the (α4)₃(β2)₂ stoichiometry. We show that the (α4)₃(β2)₂ stoichiometry and the (α4)₂(β2)₂β3 and (α4)₂(β2)₂α5 subtypes in which β3 or α5occupy the accessory positions have much higher permeability to Ca²⁺ than does (α4)₂(β2)₃ and suggest that this could be physiologically significant in triggering signaling cascades if this stoichiometry or these subtypes were found in vivo. We show that Ca²⁺ permeability is determined by charged amino acids at the extracellular end of the M2 transmembrane domain, which could form a ring of amino acids at the outer end of the cation channel. α4, α5, and β3 subunits all have a homologous glutamate in M2 that contributes to high Ca²⁺ permeability, whereas β2 has a lysine at this position. Subunit combinations or single amino acids changes at this ring that have all negative charges or a mixture of positive and negative charged amino acids are permeable to Ca²⁺. All positive charges in the ring prevent Ca²⁺ permeability. Increasing the proportion of negative charges is associated with increasing permeability to Ca²⁺.