Lobe-related concentration- and Ca2+-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel

Abstract

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca(2+) channel CaV1.2 at different CaM and Ca(2+) concentrations ([Ca(2+)]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca(2+)-dependencies. Our data show that more than one CaM molecule could bind to the CaV1.2 C-terminal tail at high [Ca(2+)]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca(2+)] in its binding to the C-terminal tail of CaV1.2, and the binding between CaM and the N-terminal tail of CaV1.2 requires high [Ca(2+)]. Our data provide new details on the interactions between CaM and the CaV1.2 channel.

The individual N‐ and C‐lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run‐down in ventricular myocytes of guinea‐pig heart

“…Some other experiments have demonstrated that CDI in the L‐type channel (Cav1.2) is governed by the binding of Ca 2+ to C‐lobe of CaM [3,8,9]. Our previous studies have also shown that C‐lobe of CaM is critical in sensing the change of [Ca 2+ ] when it binds to the C‐terminal tail of Cav1.2 [10]. However, when we observed the facilitation and inactivation of Cav1.2 activity induced by Ca 2+ ‐insensitive mutants of CaM with patch‐clamp technique, we found that the Ca 2+ ‐dependency requires both N‐ and C‐lobe [11,12].…”

“…The Ca 2+ ‐insensitive mutants of N‐lobe (N‐lobe 12 , E31A + E67A) and C‐lobe (C‐lobe 34 , S101F + E140A) were created by site‐directed mutagenesis using a Quickchange™ kit (QIAGEN) (Fig. 1) [10].…”

“…GST‐fusion CT1 (2–4 μg) was immobilized on GS‐4B and incubated in 300 μL of Tris buffer with increasing concentrations of CaM lobes (0.1, 0.35, 0.7, 1.4, 2.1, 3.5, 10 μM) for 4 h at 4 °C under agitation in the presence of different free Ca 2+ concentrations ([Ca 2+ ], ≈ free, 100 nM, 10 μM, and 2 mM) [10,16,20]. Then, the reaction mixtures were gently washed twice with the same buffer containing the non‐ionic detergent Tween 20 (0.05%, Sigma–Aldrich).…”

The Ca2+‐dependent interaction of calpastatin domain L with the C‐terminal tail of the Cav1.2 channel

“…The purification of the GST-fusion proteins was as described in the previous reports [8,12,13] As for CS L fusion protein, the GST portion of it was removed by using the PreScission Protease (GE Healthcare). All steps were performed on ice or at 4°C.…”

“…The amount of CS L and GST-fusion peptides was corrected based on the relative optical densities of 0.59 and 0.80 in reference to BSA, respectively [8,13]. …”