The agonist-induced steady-state desensitization of the N-methyl-D-aspartate (NMDA) receptor was investigated by means of whole cell patch and ...
Neuroseience Letters, 108 (1990) 88 92 Elsevier Scientific Publishers Ireland Lid,
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NSL 06570
Desensitization of N M D A receptors does not proceed in the presence of kynurenate I.V. Chizhmakov, N.I. Kiskin, A.Ya. T s y n d r e n k o and O.A. Krishtal A.A. Bogornoletz Institute o['Physiology Ukrainian Academy of Sciences, Kiev (U.S.S.R.) (Received 7 March 1989; Revised version received 11 September 1989: Accepted 11 September 1989)
Key words: Isolated neuron; Hippocampus; N-Methyl-D-aspartate receptor; Steady-state desensitization; Kynurenate; L-Aspartate: Glycine The agonist-induced steady-state desensitization of the N-methyl-D-aspartate (NMDA) receptor was investigated by means of whole cell patch and concentration clamp in isolated pyramidal neurons from rat hippocampus. When administered against a background of previously applied agonist, glycine (Gly) produced a response that was smaller than the response elicited by simultaneous application of agonist and Gly. This feature could be explained by an agonist-induced suppression of NMDA receptor sensitivity to the facilitatory action of Gly. This type of 'steady-state desensitization' did not develop when the preincubating solutions contained kynurenate (250/~M). It is proposed that NMDA receptors at all concentrations of agonist may acquire two distinct and interconverting conformations that are sensitive or insensitive to the facilitatory action of Gly. Kynurenate shifts the equilibrium between these states keeping the receptor in a Gly-sensitive conformation.
Glycine (Gly) and some analogues are known to selectively potentiate the responses of central neurons to N-methyl-D-aspartate (NMDA) and its selective agonist L-aspartate (Asp) [1, 6, 9, 15]. This modulatory effect is due to allosteric interaction between the strychnine-insensitive Gly receptor (referred to below as 'Gly* receptor') and the N M D A receptor [4, 9]. Investigations of agonist-receptor interactions at different membrane receptors (acetylcholine receptors [8, 10], n o n - N M D A glutamate receptors [12], Gly strychnine-sensitive receptors [14]) suggested the agonist-induced loss of receptor sensitivity to the agonist (desensitization). The ionic currents elicited by N M D A and its agonists in hippocampal neurons are also subject to desensitization [3, 6, 15]. With sufficiently prolonged application of agonists desensitization tends towards a certain steady-state level which is determined by the agonist concentration. Our recent data [6] led us to suspect that the N M D A receptors possibly possess a new type of steady-state desensitization, namely the agonist-induced loss of receptor sensitivity to the facilitatory action of Gly. This feature has been further studied in the present work. Correspondence." O.A. Krishtal, A.A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainain SSR, Kiev 252024, U.S.S.R. 0304-3940/90/$ 03.50 ~) 1990 Elsevier Scientific Publishers Ireland Ltd.
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Fig. 1. Responses of hippocampal neurons to Asp with I 0 pM Gly obtained with and without preincubation in the same concentration of Asp, and their dependences on Asp concentration. Here and below: bars indicate the programs of drug application, gaps in the current traces correspond to the time of preincubation of 30 s. Holding potential - 100 mV. A: responses elicited by the addition of Gly (10/~M) against a background of different concentrations of Asp (indicated at the beginning of corresponding bars). Up to 5 mM Asp alone elicited no response in this neuron. Right panel: the control response to simultaneous application of 5 mM Asp with 10/~M Gly (/max).B: dose-response relationship for the responses to simultaneous application of Asp with 10/tM Gly (Q, IN/lm~, Kd= 51 pM) and the dependence of the responses obtained by application of 10 pM Gly against a background of a preincubating Asp concentration ((3, 1A/lmax, Ka = 57/~M). Peak amplitudes of ionic currents were normalized to the amplitude of maximal response obtained by simultaneous application of 5 mM Asp with 10 pM Gly. Points represent mean values obtained in 3-4 cells; bars indicate +__S.D. Smooth curves are Langmuir isotherms with Kd values calculated to obtain the best fit using the least-squares procedure.
E x p e r i m e n t s were carried o u t on n e u r o n s isolated f r o m e n z y m a t i c a l l y p r e t r e a t e d h i p p o c a m p a l slices o f 10- to 2 0 - d a y - o l d rats. T h e p r o c e d u r e o f e n z y m a t i c t r e a t m e n t has been described elsewhere [6]. The n e u r o n s were p u l l e d o u t with the p a t c h pipette directly f r o m the t r e a t e d slice which was s e p a r a t e d a l o n g the p y r a m i d a l cell layers CA1 a n d CA3. T h e artificial intracellular s o l u t i o n c o n t a i n e d 100 m M K F a n d 30 m M Tris-C1, at p H 7.2. C o n t r o l s p e r f o r m e d in the absence o f F - ( C s O H - H 3 P O 4 130 m M , M g C I 2 - A T P 2 m M ) gave the same results. T h e n o r m a l external s o l u t i o n c o n t a i n e d (mM): N a C I 150, K C I 5, CaC12 5, H E P E S - N a O H 10, p H 7.4. T h e c o n c e n t r a t i o n c l a m p technique [13] ensured a c o m p l e t e exchange o f s o l u t i o n a r o u n d a large p y r a m i d a l n e u r o n which preserved a s u b s t a n t i a l p a r t o f the dendritic tree [12] within 15-30 ms. E x p e r i m e n t s were carried o u t at 21-24°C. A g o n i s t s were a d d e d to the salines in f o r m o f Tris salts. T h e m a j o r i t y o f experim e n t s were p e r f o r m e d with Asp; c o n t r o l s with N M D A gave the same results. T h e
90 chemicals were purchased from Sigma, except N M D A (Cambridge Research Biochemicals). Fig. IA demonstrates an experiment in which Gly in saturating concentration was applied to the cell against a background of different concentrations of Asp. For all the neurons tested (n = I1), the maximal amplitude of the response (1A) facilitated by Gly never exceeded the maximal response (lm~j to the simultaneous application of 5 mM Asp with 10 pM Gly (normalized mean _+S.D., 0.28_.+0.11, significantly different from unity, P
