The λ1 subunit of the SM L-type Ca²⁺ channel reduces Ca²⁺ influx by reducing the current amplitude and by a negative shift in the steady-state inactivation, whereas the sensitivity to the DHP agonist (-)-Bay K 8644 is not affected (Freise et al. 2000). We tested the binding and blocking action of the DHP isradipine and the BTZ diltiazem on the SM L-type Ca²⁺ channel from λ1-deficient and wild-type (WT) mice. The densities of isradipine binding sites were not significantly different at 4 °C (WT: B_max, 1.27 ± 0.1 pmol (mg protein)^-1 (mean ± S.E.M.); K_D, 0.54 ± 0.7 nM, n = 3; λ1-/-: B_max, 1.26 ± 0.17 pmol (mg protein)^-1; K_D, 0.54 ± 0.1 nM; n = 3). However, at 37 °C binding site densities were significantly reduced in λ1-/- microsomes (λ1-/-: B_max, 0.54. ± 0.16 pmol (mg protein)^-1; n = 4; WT: B_max, 1.23 ± 0.34 pmol (mg protein)^-1; n = 4). Diltiazem (10 mM) increased isradipine binding site densities to similar levels at 37 °C in WT cells (B_max, 2.39. ± 0.55 pmol (mg protein)^-1; n = 4) and λ1-/- cells (B_max, 2.02 ± 0.62 pmol (mg protein)^-1; n = 4). This effect was observed in the absence or presence of Ca²⁺. Apparently, in the absence of λ1 the high-affinity calcium channel blocker binding site within α1S is destabilized at 37 °C; however, binding can be restored in the presence of diltiazem. To test whether the altered binding is reflected in an altered channel sensitivity to these blockers, L-type Ca²⁺ channel currents were measured in myotubes in the whole-cell configuration of the patch-clamp technique as described previously (Freise et al. 2000). Ca²⁺ currents (10 mM Ca²⁺ as charge carrier) were activated by step depolarisations from -90 mV to various test potentials in the absence and presence of increasing concentrations of isradipine and diltiazem. The apparent IC₅₀ values for Ca²⁺ current inhibition at +20 mV by isradipine and diltiazem were 177 nM and 102 mM (WT cells) and 455 nM and 383 mM (λ1-/- cells), respectively. Accordingly, the current was blocked by 58 % (WT cells) and 38 % (λ1-/- cells) in the presence of 50 nM isradipine and 10 mM diltiazem. The 58 % inhibition observed in WT cells apparently agrees with the 63 % inhibition expected if additive block by isradipine and diltiazem is assumed, whereas in λ1-/- cells, an additive block by only 23 % was expected. Apparently, the block in in λ1-/- cells is more than additive as expected from the binding experiments. From these data, we conclude that the λ1 subunit of the SM L-type Ca²⁺ channel influences the binding and the blocking effects of DHPs and BTZs.

This work was supported by the Deutsche Forschungsgemeinschaft.

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