Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis

The pores of glutamate receptors and K+ channels share sequence homology su ggesting a conserved secondary structure. Scanning mutagenesis with substit ution of alanine and trytptophan in GluR6 channels was performed based on t he structure of KcsA. Our assay used disruption of voltage-dependent polyam ine block to test for changes in the packing of pore-forming regions. Alani ne scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A pel-iodic pattern from F575 to M589 aligne d with the pore helix in KcsA, whereas a cluster of sensitive positions aro und Q590. a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D56T to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 3T positi ons and a phl dissociation constant for Q590W Molecular modeling with KcsA coordinates showed that GluR6 pol e helix mutants disrupting polyamine bloc k pack against M1 and M2, and are not exposed in the ion channel pore. In t he selectivity filter tryptophan creates an aromatic cage consistent with t he pM dissociation constant for Q590W A scan with glutamate substitution wa s used to map the cytoplasmic entrance to the pore based on charge neutrali zation experiments, which established that E594 was uniquely required fur h igh affinity polyamine block. In E594Q mutants, introduction of glutamate a t positions S593-L600 restored pol) amine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that t he pore region of glutamate receptors contains a helix and pore loop analog ous to that found in K+ channels. At the cytoplasmic entrance of tile chann el, a negatively charged amino acid, located in an extended loop with solve nt-exposed side chains, is required for high affinity polyamine block and p robably attracts cations via a through space electrostatic mechanism.