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 dihydropyridinesensitive 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.calcium͞calmodulin-dependent protein kinase type II ͉ calcineurin ͉ dihydropyridine ͉ excitotoxicity

Inhibitory Phosphorylation of GSK-3 by CaMKII Couples Depolarization to Neuronal Survival

“…Furthermore, kinetic assays, with CaMKII␤ as kinase and GSK-3␣ or GSK-3␤ as substrate, were conducted to assess the kinetics of phosphorylation reactions as described previously (21) , respectively. As compared with the reported data for CaMKII phosphorylation of autocamtide (66) and for PKA phosphorylation of GSK-3␤ (21), the relatively low K m value for the in vitro phosphorylation of GSK-3 by CaMKII is consistent with both GSK-3␣ and GSK-3␤ acting as physiological substrates of CaMKII␤. We conclude from these data that CaMKII␤ phosphorylates GSK-3␣ and GSK-3␤ both in vivo and in vitro.…”

The Distinct Roles of Calmodulin and Calmodulin Kinase II in the Reversal of Run-Down of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…In addition to the IQ motif, another three CaM-binding sites, the preIQ region in the C-terminal tail (22,23), the NSCaTE motif in the N-terminal of Cav1(α 1 ) Ca 2+ channels (24), and I-II linker (26), have been identified. The complexes formed by CaM and each of these motifs might allow CaM to perform the two opposing processes of CDF and CDI.…”

“…These phenomenons suggested that the endogenous CaM and CaMKII were perhaps washed out gradually, and they have different and closely related effects in the regulation of Ca 2+ channels. As mentioned above, there are at least four CaMbinding sites (22 -24, 26) and several CaMKII phosphorylation sites (26,34) in the Cav1.2 Ca 2+ channel. Therefore it is possible that CaMKII phosphorylates multiple sites and affects the multiple binding sites of CaM as inferred from Fig.…”

“…We recently found that the threonine site Thr1603 was phosphorylated by CaMKII. Erxleben et al (26) have reported that CaMKII phosphorylates Ser439 in the I-II loop (which contains a CaM-binding site) and Ser1517 (Ser1516 in guinea pig), thereby promoting mode 2 activity in the rabbit cardiac Cav1.2 channel. Lee et al have also reported that CaMKII phosphorylates Ser1512 and Ser1570 (Ser1517 / Ser1516 and Ser1575 / Ser1574 in rabbit / guinea-pig cardiac type) in the C-terminal tail of the rabbit smooth muscle Cav1.2 channel and thereby promotes voltage-dependent facilitation (34).…”

Swapping the I-II intracellular linker between L-type CaV1.2 and R-type CaV2.3 high-voltage gated calcium channels exchanges activation attributes

“…[18][19][20][21] In addition, residues located at the cytoplasmic end of IS6 have been reported to affect channel opening. [22][23][24] These residues seem to coincide with the bundle crossing region where S6 from other domains come together (reviewed by Hering, et al). 6 Thus, it appears coherent that the I-II linker adds a component to the channel gating machinery as it is physically connected to IS6 of the voltage-dependent ion channel family.…”