Modulation of neuronal responses to AMPA by membrane cholesterol

M.A. Simmonds & F. Fariba.
Department of Pharmacology, School of Pharmacy, 29/39 Brunswick Square, London, WC1N 1AX.

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Simmonds MA
Fariba F

The methodological approach used previously to study the influence of membrane cholesterol on the responsivity of GABA_Areceptors in acutely dissociated rat hippocampal neurones (Sooksawate et al., 2001) has now been applied to AMPA receptors in the same neuronal population. Current responses to 1s local applications of AMPA typically showed a rapid rise to peak followed by a rapid decay to a plateau. About 20% of neurones were rejected for study due to lack of a clear plateau (either minimal or complete decay). The response decay in the continued presence of AMPA has generally been attributed to receptor desensitization (Fleck et al., 1996).

Neuronal membrane cholesterol levels were altered by means of the cholesterol carrier methyl-ß-cyclodextrin. Depletion to 62 ± 2.3% control increased the potency of AMPA 2.5-fold with a leftward shift of the log. concentration - peak response curve without change in the maximum response (EC₅₀values: control 24.1 ± 0.56 µM, n=18; depleted 9.8 ± 0.43 µM, n=16 (mean ± s.e. mean)). In contrast, enrichment with cholesterol to 206 ± 4.8% control reduced the maximum of the AMPA log. concentration - peak response curve to 58.5 ± 6.39 % control and increased the EC₅₀ value to 57.5 ± 1.02 µM (n=19).

Decay from the peak response to a plateau was concentration-related and reached a maximum at 100 µM AMPA and above. The maximum % decay was 78.± 2.9 (18) in control neurones, 65.1 ± 3.6 (16) in cholesterol-depleted neurones and 87.2 ± 3.5 (19) in cholesterol-enriched neurones. These values were significantly different from each other (ANOVA + Dunnett's test, P<0.05). In some neurones, a single exponential time constant (_D) for the decay from peak was also measured. The values obtained were 47.3 ± 0.09 ms (n=6) in control neurones, 90.6 ± 0.29 ms (n=8) in depleted neurones and 23.4 ± 0.18 ms (n=11) in enriched neurones.

The competitive AMPA antagonist NBQX (Parsons et al., 1994) at 6 µM caused a 4.8-fold shift to the right of the log. concentration - peak response curve for AMPA (EC₅₀ values: before NBQX 24.7 ± 0.89 µM; after NBQX 119.4 ± 1.74 µM, n=13) without depression of the maximum response. The % decay from peak response to plateau maintained the same dependency on AMPA concentration in the presence and absence of NBQX. The non-competitive antagonist GYKI52466 (Donevan et al., 1993) at 15 µM depressed the maximum of the log. concentration - peak response curve to 57.6 ± 0.88 % control (n=17), with an increase in EC₅₀ from 24.0 ± 0.69 µM to 34.0 ± 1.52 µM. The dependency on AMPA concentration of the % decay from peak response to plateau was shifted by GYKI52466 to 2.8-fold higher concentrations of AMPA but the maximum % decay was unchanged by the antagonist.

The increased potency of AMPA in cholesterol-depleted neurones and the reduction in peak current responses to AMPA in cholesterol-enriched neurones might result from corresponding changes in receptor affinity and/or from the concomitant changes in rate and degree of decay of the current from the peak. The decay mechanism appears to depend on an action of AMPA that is distinct from that which evokes the current response since it was unaffected by NBQX, although it was attenuated by GYKI52466.

These effects of membrane cholesterol on AMPA receptors are distinct from those previously reported for GABA_A receptors.

Donevan, S.D. et al., (1993) Neuron 10, 51-59.
Fleck, M.W. et al., (1996) J Neurophysiol 75, 2322-2333.
Parsons, C.G. et al., (1994) Neuropharmacology 33, 589-604.
Sooksawate, T. et al., (2001) Neuropharmacology 40, 178-184