Effects of 5'-alkyl-benzothiadiazides on (R, S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor biophysics and synaptic responses

Alkyl-substituted benzothiadiazides (BTDs) were tested for their effects on (R,S)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. In excised patches, the 5-ethyl derivative “D1” blocked the desensitization of AMPA receptor currents during prolonged application of glutamate (EC50 ,3 6M), and it slowed deactivation of responses elicited by 1-ms glutamate pulses greater than 10fold. [ 3 H]Fluorowillardiine binding to rat synaptic membranes was increased by D1 by a factor of 3.6 (EC50 ,1 7M) with a Hill coefficient near 2. In hippocampal slices, the compound reversibly increased excitatory postsynaptic currents and field excitatory postsynaptic potentials (EPSPs) with thresholds around 10 M. The size of the alkyl substituent influenced both the potency and nature of the drug effect on synaptic currents: 5-methyl compounds had a 2-fold greater effect on response amplitude than on response duration, whereas 5-ethyl compounds like D1 caused greater increases in duration than amplitude. In tests with recombinantly expressed AMPA receptor subunits, D1 preferred the glutamate receptor (GluR) subunit GluR4 flip (0.64 M) over GluR4 flop (5.3 M); similar affinities but with smaller flip-flop differences were obtained for GluR1 through 3. These results show that D1 and congeners are significantly more potent than the parent compound IDRA-21 and that they differ in two fundamental aspects from cyclothiazide, the most widely studied BTD: 1) D1 markedly increases the agonist affinity of AMPA receptors and 2) it has immediate and large effects on field EPSPs. The large gain in potency conferred by alkyl substitution suggests that the 5 substituent is in intimate contact with the receptor, with the size of the substituent determining the way in which receptor kinetics is changed. AMPA-type glutamate receptors are abundant throughout the brain and account for much of the transmission occurring at excitatory synapses. Ito et al. (1990) made the seminal observation that the current through these receptors can be enhanced by the nootropic compound aniracetam. The drug did not influence other types of glutamate receptors, and it had no evident effect on AMPA receptors in the absence of glutamate. Other compounds were subsequently discovered that “up-modulate” or “potentiate” AMPA receptor function in a similar manner, including diazoxide (Yamada and Roth