Cyclosporin and Timothy syndrome increase mode 2 gating of CaV1.2 calcium channels through aberrant phosphorylation of S6 helices

Abstract

Calcium channels in the plasma membrane rarely remain open for much more than a millisecond at any one time, which avoids raising intracellular calcium to toxic levels. However, the dihydropyridine-sensitive calcium channels of the CaV1 family, which selectively couple electrical excitation to endocrine secretion, cardiovascular contractility, and neuronal transcription, have a unique second mode of gating, "mode 2," that involves frequent openings of much longer duration. Here we report that two human conditions, cyclosporin neurotoxicity and Timothy syndrome, increase mode 2 gating of the recombinant rabbit CaV1.2 channel. In each case, mode 2 gating depends on a Ser residue at the cytoplasmic end of the S6 helix in domain I (Ser-439, Timothy syndrome) or domain IV (Ser-1517, cyclosporin). Both Ser reside in consensus sequences for type II calmodulin-dependent protein kinase. Pharmacologically inhibiting type II calmodulin-dependent protein kinase or mutating the Ser residues to Ala prevents the increase in mode 2 gating. We propose that aberrant phosphorylation, or "phosphorylopathy," of the CaV1.2 channel protein contributes to the excitotoxicity associated with Timothy syndrome and with chronic cyclosporin treatment of transplant patients.

Fig. 1.

Inhibition by cyclosporine (CsA) of the calcium/calmodulin-dependent protein phosphatase IIB calcineurin results in an increase of long, mode 2 openings of native l-type channels in neurons and pituitary cells. Shown are representative records from cell-attached patches at 0 mV and open time histograms for all open events from one patch for dissociated primary embryonic rat cortical neurons (A), neurons treated with 20 μM cyclosporin (B), GH₄C₁ cells treated with 20 μM cyclosporin (C), and GH₄C₁ cells treated with 20 μM cyclosporin and 10 μM KN-62 (D). The dashed lines in the channel-open time histograms are maximum-likelihood fits by the sum of two exponentials, which represent short (τo₁) and long (τo₂) open times. (E and F) Summaries of the values for the frequency of mode 2 openings from the indicated cell type and treatment. Con, control.

Fig. 2.

Spontaneous mode 2 gating of recombinant rabbit cardiac CaV1.2 channels. Shown are representative records and open time histograms for all events from one patch for wild-type channels (A), the missense mutation G436R found in Timothy syndrome (B), inhibition of CaMKII with 10 μM KN-62 (C), or the double mutation G436R, S439A (D). (E) Summary of the values for the long open times, τo₂, as well as the frequency of mode 2 openings from the indicated number of patches expressing each construct. wt, wild type.

Fig. 3.

CaV1.2 peptides are phosphorylated by purified CaMKII in vitro. (A) Time dependence of ³²P incorporation into designated peptides. (B) Concentration dependence of initial rate of phosphorylation. (C and D) MALDI-TOF MS/MS spectra of the G436R IS6 peptide (C) and the native Ser-1517 IVS6 peptide (D) with only diagnostic y series ions labeled for simplicity. (C) The presence of y₈ and y₉ ions exhibiting the loss of H₃PO₄ identifies the site of phosphorylation to Ser-8. (D) Similarly, the presence of a y₉ ion with the loss of H₃PO₄ locates the site of phosphorylation to Ser-7. RxxS of each peptide form the CaMKII consensus sequence.

Fig. 4.

Mutation of Ser-1517 at the cytoplasmic end of IVS6 decreases the frequency of mode 2 openings. Shown are representative records and open time histograms for all events from one patch for S1517A (A), S1517A treated with 20 μM cyclosporin (B), and S1517A treated with 100 nM (−)Bay K8644 (C). (D) Summary of the values for the long open times, τo₂, as well as the frequency of mode 2 openings from the indicated number of patches under each condition. CsA, cyclosporin; Bay K, Bay K8644.

Fig. 5.

Pivotal role of Ser phosphorylation as a regulatory mechanism of CaV1.2 mode 1/mode 2 gating. (A) Summary of mutations and conditions favoring mode 1 and mode 2 gating. (B) Location of critical amino acids affecting mode 1/mode 2 gating. Mutant G436R creates a CaMKII site, and the phosphorylation of Ser-439 favors mode 2 gating. Mutant S1517A abrogates the effect of protein phosphatase IIB (PP2B) inhibition by cylosporin to promote mode 2 gating. Mutant T1066Y prevents Bay K8644 binding and its promotion of mode 2 gating. Mutant S1142A abrogates the effect of the dihydropyridine by an allosteric mechanism involving Ser-1517 (18). (C) Three-dimensional model of the rabbit CaV1.2 α₁ subunit’s S5–Pore–S6 segments based on the crystal structure of the Kcs K channel. Details are as in ref. . The locations of the Ser residues whose mutation to Ala affect the gating modes are highlighted in yellow, with gamma oxygen in red. Location of basic amino acids required for phosphorylation of Ser-439 and Ser-1517 by CaMKII are in deep blue; Thr-1066 critical for dihydropyridine binding is in light blue, and the dihydropyridine agonist (−)Bay K8644 is in green. Note that Ser-439 and Ser-1517 are similarly positioned at the inner mouth of the channel.