A molecular link between inward rectification and calcium permeability of neuronal nicotinic acetylcholine alpha 3 beta 4 and alpha 4 beta 2 receptors

Many nicotinic acetylcholine receptors (nAChRs) expressed by central neuron s are located at presynaptic nerve terminals. These receptors have high cal cium permeability and exhibit strong inward rectification, two important ph ysiological features that enable them to facilitate transmitter release. Pr eviously, we showed that intracellular polyamines act as gating molecules t o block neuronal nAChRs in a voltage-dependent manner, leading to inward re ctification. Our goal is to identify the structural determinants that under lie the block by intracellular polyamines and govern calcium permeability o f neuronal nAChRs. We hypothesize that two ring-like collections of negativ ely charged amino acids (cytoplasmic and intermediate rings) near the intra cellular mouth of the pore mediate the interaction with intracellular polya mines and also influence calcium permeability. Using site-directed mutagene sis and electrophysiology on alpha(4)beta(2) and alpha(3)beta(4) receptors expressed in Xenopus oocytes, we observed that removing the five negative c harges of the cytoplasmic ring had little effect on either inward rectifica tion or calcium permeability. However, partial removal of negative charges of the intermediate ring diminished the high-affinity, voltage-dependent in teraction between intracellular polyamines and the receptor, abolishing inw ard rectification. In addition, these non-rectifying mutant receptors showe d a drastic reduction in calcium permeability. Our results indicate that th e negatively charged glutamic acid residues at the intermediate ring form b oth a high-affinity binding site for intracellular polyamines and a selecti vity filter for inflowing calcium ions; that is, a common site links inward rectification and calcium permeability of neuronal nAChRs. Physiologically , this molecular mechanism provides insight into how presynaptic nAChRs act to influence transmitter release.