The amino acid residue at sequence position 5 in the conantokin peptides partially governs subunit-selective antagonism of recombinant N-methyl-D-aspartate receptors

Whole cell voltage clamp recordings mere performed to assess the ability of conantokin-G: (con-G), conantokin-T (con-T), and a 17-residue truncated fo rm of conantokin-R (con-R[1-17]) to inhibit N-methyl-n-aspartate (NMDA)evok ed currents in human embryonic kidney 293 cells transiently expressing vari ous combinations of NR1a, NR1b, NR2A, and NR2B receptor subunits. Con-T and conR[1-17] attenuated ion currents in cells expressing NR1a/ NR2A or NR1a/ NR2B, Con-G,did not affect NMDA-evoked ionic currents in cells expressing N R1a/NR2A, but it showed inhibitory activity in cells expressing NR1a/NR2B r eceptors and the triheteromeric combination of NR1a/ NR2A/NR2B. An Ala-rich con-G analog, con-G[Q6G/gamma 7K/ N8A/gamma 10A/gamma 14A/K15A/S16A/N17A] (Ala/con-G, where gamma is Gla), in which all nonessential amino acids were altered to Ala residues, manifested subunit specificity similar to that of con-G, suggesting that the replaced residues are not responsible for selec tivity in the con-G framework. A sarcosine-containing con-T truncation anal og, con-T[1-9/ G1Src/Q6G], inhibited currents in NR1a/NR2A and NR1a/ NR2B r eceptors, eliminating residues 10-21 as mediators of the broad subunit sele ctivity of con-T, In contrast to the null effects of con-G and Ala/con-G at a NR1a/NR2A-containing receptor, some inhibition (similar to 40%) of NMDA- evoked currents was effected by these peptides in cells expressing NR1b/NR2 A. This finding suggests that the presence of exon 5 in NR1b plays a role i n the activity of the conantokins, Analysis of various conantokin analogs d emonstrated that Leu(5) of con-G is an important determinant of conantokin selectivity, Taken as a whole, these results suggest that the important mol ecular determinants on conantokins responsible for NMDA receptor activity a nd specificity are discretely housed in specific residues of these peptides , thus allowing molecular manipulation of the NMDA receptor inhibitory prop erties of the conantokins.