The role of hydrophobic interactions in binding of polyamines to non NMDA receptor ion channels
Introduction
Although it is now widely recognized that ion channel block by cytoplasmic polyamines underlies the biphasic current voltage relationship characteristic of AMPA and kainate receptors assembled from subunits with Gln residues at the Q/R site in the pore forming region (Verdoorn et al., 1991, Bowie and Mayer, 1995, Donevan and Rogawski, 1995, Isa et al., 1995, Kamboj et al., 1995, Koh et al., 1995), surprisingly little is known concerning the molecular mechanism(s) and binding site(s) underlying the effects of these aliphatic cations. Spermine and spermidine differ from the majority of conventional ion channel blockers in that they are long rod shaped molecules with multiple charged NH2 groups interspersed with hydrophobic CH2 groups. Hence it is conceivable that the binding site for polyamines could involve multiple amino acid residues rather than a discrete locus. Indeed, the alternating NH2 and CH2 groups in polyamines would be expected to preferentially interact with charged and hydrophobic amino acid side chains, respectively, similar to the active site of bacterial polyamine binding proteins (Sugiyama et al., 1996). Such a mechanism of action is likely to occur for the action of many drugs which block ion channel pores, including quaternary and bis-quaternary ammonium alkylamine block of K-channels (Armstrong, 1971, Miller, 1982, Choi et al., 1993) and n-alkyl diamine block of NMDA receptors (Subramaniam et al., 1994), but has not been considered before for the highly charged endogenous polyamines which block inward rectifier K-channels, non-NMDA subtype glutamate receptors and neuronal nicotinic acetylcholine receptors (Nichols and Lopatin, 1997, Williams, 1997, Haghighi and Cooper, 1998). In an attempt to get information concerning the influence of the methylene groups in polyamines on their affinity for strongly rectifying glutamate receptor channels we have performed a systematic analysis of the structure-activity relationships for block of kainate receptors assembled from GluR6(Q). Our results suggest that much of the binding energy for channel block by polyamines comes from hydrophobic interactions. In addition, analysis of block by diamines with identical numbers and seperation of NH2 groups but different numbers of external CH2 groups suggests that displacement of permeant ions from the pore region may contribute to the voltage dependence of block by polyamines.
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Transfection and recording techniques
Homomeric GluR6(Q) glutamate receptor channels were transiently expressed in HEK 293 cells as described previously (Bowie and Mayer, 1995, Bähring et al., 1997). Co-transfection of glutamate receptor cDNA (150 ng 35 mm−1 dish) with cDNA for the S65T mutant of green fluorescent protein (ratio 1:1), both gifts from Dr P. Seeburg, Department of Molecular Neurobiology, Max-Planck-Institute for Medical Research, Heidelberg, allowed the identification of transfected cells during experiments by
Washout of block by endogenous polyamines
Because, in the absence of chelators such as ATP, block by endogenous polyamines shows gradual washout for up to 15 min or more following formation of outside out patches (Fig. 1), it was critical in the present experiments to ensure that the responses recorded reflected the action of experimentally added polyamines, and not block by residual endogenous spermine and spermidine (Bowie and Mayer, 1995, Bähring et al., 1997). This was achieved by analyzing multiple GV plots recorded at intervals
Discussion
Our results suggest that much of the binding energy for block of glutamate receptor channels by cytoplasmic polyamines arises from hydrophobic interactions. This was unexpected given the existence in spermine of multiple ionized ammonium groups and the strong voltage dependence of polyamine block (Bowie and Mayer, 1995, Bähring et al., 1997). However, several factors combine to complicate any simple analysis and interpretation of the blocking action of polyamines on ion channels. First is the
Acknowledgements
We thank Professors Shirahata and Nakanishi for the gifts of compounds; Dr P. Seeburg for plasmids; and C. Glasser for preparation of cDNAs.
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These authors contributed equally.
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Present address. Institute für Neurale Signalverarbeitung, Zentrum für Molekulare Neurobiologie Uinversitäts-Krankenhaus Eppendorf, Martinistraße 52, 20246 Hamburg, Germany.