The NMDA receptor NR1 subunit intracellular C-terminus has two binding sites for calmodulin: the C0 and C1 regions (Ehlers et al. 1996). Previously we have shown that the NMDA receptor mean open time is reduced by occupation of the high-affinity C1 region by calmodulin (Rycroft & Gibb, 1999). Here we studied the effect of calmodulin binding at the low-affinity C0 region using α-actinin, which competes with calmodulin for the C0 region and is thought to be responsible for anchoring synaptic NMDA receptors to the actin cytoskeleton (Wyszynski et al. 1997).

NMDA receptors, activated by 100 nM NMDA and 10 µM glycine, were studied in outside-out membrane patches isolated from granule cells in the dentate gyrus of hippocampal slices prepared from humanely killed 10-day-old rats. 800 nM free calcium, 800 nM free calcium plus 800 nM active calmodulin, or 800 nM free calcium plus 800 nM active calmodulin plus 2.5 µM α-actinin were applied to the intracellular membrane surface. At these concentrations, calmodulin binding to the C0 region is expected to be reduced by α-actinin by approximately 80 %, while binding to the high-affinity C1 region may be unaffected.

The NMDA receptor single-channel conductance was not significantly affected by the application of calmodulin, or calmodulin plus α-actinin (analysis of variance, P > 0.05, n = 6, 7 and 6 patches, respectively). Likewise, the NMDA receptor shut times were unaffected by these treatments. However, the NMDA receptor mean open time (mean ± S.E.M. at -60 mV) was 4.14 ± 0.6 ms in 800 nM calcium, 2.93 ± 0.4 ms in 800 nM calmodulin and 1.84 ± 0.4 ms in calmodulin plus α-actinin. The voltage dependence of the mean open time (between -80 and -20 mV) was not significantly different (analysis of covariance, P > 0.05) between the treated groups increasing e-fold for every 47 mV depolarisation. However, the mean open time was significantly (analysis of covariance, P < 0.001) reduced by calmodulin and suffered a further significant reduction by the presence of α-actinin.

These results may suggest that α-actinin binding at C0 produces a further reduction of channel open time, additional to that produced by calmodulin binding at C1. Alternatively, some form of negative co-operativity may exist between calmodulin binding at the two regions and when this is prevented by displacement of calmodulin from the C0 site, the full effect of calmodulin binding at C1 is unmasked.This work was supported by the MRC.

Ehlers, M.D., Zhang, S., Bernhadt, J.P. & Huganir, R.L. (1996). Cell 84 (5), 745-755.

Rycroft, B. & Gibb, A.J. (1999). J. Physiol. 518.P, 137P.

Wyszynski, M., Lin, J., Rao, A., Nigh, E., Beggs, A.H., Craig, A.M. & Sheng, M. (1997). Nature 385, 439-442.