Regulation of Cav1.2 Current: Interaction with Intracellular Molecules

Regulation of CaV1.2 Current: Interaction With Intracellular Molecules

Takeshi Kobayashi1,*, Yoichi Yamada1, Mitsuhiro Fukao1, Masaaki Tsutsuura1, and Noritsugu Tohse1 1Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan

Received January 10, 2007

Abstract. CaV1.2 (α1c) is a pore-forming subunit of the voltage-dependent L-type calcium channel and is expressed in many tissues. The β and α2/δ subunits are auxiliary subunits that affect the kinetics and the expression of CaV1.2. In addition to the β and α2/δ subunits, several molecules have been reported to be involved in the regulation of CaV1.2 current. Calmodulin, CaBP1 (calcium-binding protein-1), CaMKII (calcium/calmodulin-dependent protein kinase II), AKAPs (A-kinase anchoring proteins), phosphatases, Caveolin-3, β2-adrenergic receptor, PDZ domain proteins, sorcin, SNARE proteins, synaptotagmin, CSN5, RGK family, and AHNAK1 have all been reported to interact with CaV1.2 and the β subunit. This review focuses on the effect of these molecules on CaV1.2 current.

Keywords: CaV1.2, calcium, channel, current

Introduction on the molecules that interact with CaV1.2 and the β subunit. Previous investigators have shown that the voltage- dependent calcium channel is a multi-subunit complex Calmodulin comprising a pore-forming subunit (α subunit) and regulatory/auxiliary subunits (β, α2/δ, and γ) (1). CaV1.2 Calmodulin (CaM) is a ubiquitously expressed cal- is one of the α subunits (α1c) of the voltage-dependent cium-binding protein. CaM is thought to be involved in L-type calcium channel and is expressed in many tissues calcium-dependent inactivation (CDI), calcium-depen- including heart, ovary, pancreas, brain, and vascular dent facilitation (CDF), and the rundown phenomenon smooth muscle (2). In addition to the β and α2/δ of CaV1.2 current (2, 3). Although several models have subunits, which affect the kinetics and expression of been proposed, the exact mechanism of the regulation of CaV1.2, many molecules have been reported to be CaV1.2 current by CaM remains unclear (2). The A, C or involved in the regulation of CaV1.2. Previous binding CB, and IQ regions within the C terminal tail of CaV1.2 studies revealed that some of these molecules could have been reported to bind CaM (2, 4) (see Fig. 1). bind multiple regions of CaV1.2 (e.g., CaM can bind to Synthetic peptides or YFP-tagged proteins containing the N terminal tail, the I-II linker, and the C terminus). these regions bind CaM with different affinities and with Several different molecules bind the same region of varying degrees of dependence upon calcium (4, 5). CaV1.2 (e.g., CaM, CaBP1, and CaMKII all bind to the Using chimeric molecules comprising CaV1.2 and CaM, N terminal tail of CaV1.2), indicating that these Mori et al. (6) showed that a single molecule of CaM molecules may compete with each other to regulate the binds to CaV1.2. High-resolution crystal structure kinetics of CaV1.2 current. Therefore, we must consider analysis revealed that the calcium/CaM-IQ region all of these molecules to gain a better understanding of complex forms at least two conformations (7). CaM can the function of CaV1.2 current. In this review, we focus form several different structures depending on the particular binding partner, e.g., myosin light chain, *Corresponding author. [email protected] calcium/CaM-dependent kinase kinase peptide, adenylyl Published online in J-STAGE: doi: 10.1254/jphs.CR0070012 cyclase exotoxin, and so on (2). CaM also can bind to the N terminal tail and I-II linker of CaV1.2 (4, 8). These Invited article reports led us to speculate that CaM may form a variety

1 2 T Kobayashi et al Molecules Interacting With Cav1.2 3 of structures in the regulation of CaV1.2 current. The calcium/CaM. CaMKII can bind to the N terminus, the rundown phenomenon is usually observed within 1 – 3 I-II, II-III, and III-IV linker regions and the C terminus min of patch excision in the inside-out patch-clamp of CaV1.2 in the presence of calcium/CaM and ATP. mode. This rundown phenomenon is prevented by the They concluded that the interaction of CaMKII with the application of CaM and ATP to the intracellular solution C terminus of CaV1.2 is essential for CDF. In contrast, within 1 min of patch excision. Application of either Grueter et al. (13) reported that CaMKII binds to the C CaM or ATP alone could not restore channel activity (3). terminus of the β2a subunit and that phosphorylation of This report indicated that ATP plays an important role in Thr498 in the β2a subunit is essential for modulation of the regulation of CaV1.2 channel activity by CaM. Future CDF by CaMKII. Recently, Lee et al. (14) reported that experiments will be needed to reveal the exact role of VDF of the CaV1.2 current requires the phosphorylation ATP in the relationship between CaV1.2 and CaM. of Ser1512/Ser1570 of CaV1.2 (Ser1535/Ser1593 of human CaV1.2) by CaMKII. Mutation of Thr498 in the CaBP1 β2a subunit to Ala did not prevent VDF. These reports led us to speculate that the mechanism of signaling through Calcium-binding protein-1 (CaBP1) is a member of CaMKII, which mediates CDF and VDF, may involve the EF-hand superfamily of proteins and is closely different parts of the channel sequence or a more related to CaM (9). CaBP1 is expressed in the brain, complicated mechanism. retina, and gastrointestinal smooth muscles, but not in cardiomyocytes (9, 10). Zhou et al. (8, 11) showed that AKAPs CaBP1 can bind to the N terminus, the III-IV linker region, and the C-IQ region at the C terminus of CaV1.2. A-kinase anchoring proteins (AKAPs) contain a CaBP1 can compete with CaM for interaction with the common structural motif that tethers PKA through IQ region. In cells transfected with CaV1.2, CaBP1 interaction with a dimer of kinase regulatory subunits. prevented CDI and caused CDF of the CaV1.2 current, AKAPs bind several PKA substrates and regulate PKA- although CaM promotes strong CDI of the CaV1.2 dependent phosphorylation of these substrates (15). current. Deletion of the N terminus of CaV1.2 abolished Although more than 70 different AKAPs have been dis- the effects of CaBP1 on CDI, indicating that the covered, we focus here on AKAP15/18 and MAP2B, interaction of CaBP1 with the N terminus of CaV1.2 which have been reported to associate with CaV1.2. is essential for CaV1.2 modulation by CaBP1. The AKAP15 and AKAP18 were independently identified significance of the interaction of CaBP1 with the III-IV as 15- and 18-kDa AKAP by Gray et al. (16) and linker region of CaV1.2 remains unclear. Fraser et al. (17), respectively. However, amino acid alignment has revealed that these two proteins are CaMKII identical. Thus, this AKAP will be referred to as AKAP15/18 in this review. AKAP15/18 is expressed Calcium/CaM-dependent protein kinase II (CaMKII), in many tissues, including heart, brain, skeletal muscle, a multifunctional Ser/Thr protein kinase, has been and lung (16, 17). Previous studies reported that reported to be involved in CDF and voltage-dependent AKAP15/18 contributes to cAMP- and PKA-dependent facilitation (VDF) of CaV1.2 current (12 – 14). Hudmon augmentation of the CaV1.2 current (17, 18). Hulme et al. (12) reported that CaMKII binds to the N terminus et al. (18) reported that AKAP15/18 interacts directly and III-IV linker region of CaV1.2 in the presence of with the distal C terminus of CaV1.2 via a leucine zipper (LZ)-like motif. Disruption of this interaction using competing peptides inhibits regulation of the CaV1.2 current by the β-adrenergic receptor in rat ventricular Fig. 1. Alignment of the deduced amino-acid sequence of human CaV1.2 (UniProt accession number Q13936). The bars show the myocytes. AKAP15/18 also has a myristoylation site at putative binding regions or fragments of CaV1.2 that can interact the N terminal glycine residue and two palmitoylation with CaM, CaBP1, CaMKII, AKAP15/18, PP2A, NIL-16, sorcin, sites at Cys4 and Cys5, which are required for localiza- Sx1A, SNAP-25, Syt1, and CSN5. Some confusion over the tion to the plasma membrane (17). AKAP15/18 mutated numbering of the amino acid sequence of these molecules has arisen because different investigators have described channels from at these three residues was unable to localize to the different species and/or different splice variants. To avoid this periphery of the cell and had no effect on CaV1.2 current confusion, we have used the amino acid residue numbering of the in response to the application of a cAMP analog. These binding region/fragments based on the amino-acid sequence of reports led us to speculate that AKAP15/18 interacts human CaV1.2 (Q13936). Black shading indicates putative domain I (amino acid residues 125 – 405), domain II (525 – 753), domain III reversibly with CaV1.2 at the plasma membrane. (901 – 1186), and domain IV (1240 – 1524) of CaV1.2. Microtubule-associated protein 2 (MAP2) is a neuron- 4 T Kobayashi et al specific AKAP. Davare et al. (19) reported that MAP2b, PDZ domain proteins a splice variant of MAP2, binds directly to CaV1.2. The relative contribution of AKAP15/18 and MAP2 to It has been shown that calcium influx via postsynaptic CaV1.2 phosphorylation in neurons remains unclear. L-type calcium channels activates the transcription factor cAMP response element-binding protein (CREB) Phosphatases and CRE-dependent transcription (30, 31). CaV1.2 contains a class 1 PDZ domain-binding carboxy- As mentioned above, the anchoring of PKA by CaV1.2 terminal motif, VSXL, that has been shown to interact is essential for efficient phosphorylation of the latter. with the PDZ domain (31). Weick et al. (30) reported Since phosphorylated proteins are dephosphorylated by that interactions between the CaV1.2 subunit and PDZ phosphatases, it is reasonable to hypothesize that a domain proteins are required to activate CREB phosphatase is localized at or near CaV1.2. Davare et al. efficiently in hippocampal neurons. NIL-16 (neuronal (20) reported that protein phosphatase 2A (PP2A) binds interleukin-16 precursor protein) (32) is the only PDZ directly to the C terminus of CaV1.2. A co-immuno- domain protein that is known to interact with CaV1.2. precipitation analysis has shown that neither protein Because NIL-16 is expressed within the hippocampus phosphatase 1 (PP1) nor protein phosphatase 2B (PP2B (32), the interaction between CaV1.2 and NIL-16 is or calcineurin) binds to CaV1.2. Hall et al. (21) reported proposed to be important for efficient signaling to CREB that a stretch of amino acid residues from 1958 to in hippocampal neurons (30). However, co-immuno- about 2000 of rabbit CaV1.2 (2011-2053 of the human precipitation experiments examining potential interac- sequence) might be crucial for PP2A binding. However, tions between CaV1.2 and NIL-16 in neurons have thus the results of electrophysiological studies are not always far been unsuccessful, although a GST pull-down assay consistent with those of binding studies. Dialysis with provided evidence for direct interaction of these two the catalytic subunits of PP2A, but not PP1, resulted in a proteins (32). decrease in the L-type calcium current in native cardiomyocytes (22). In contrast, inhibitor-2, a specific Sorcin inhibitor of PP1, increased the L-type calcium current, whereas fostriecin, a specific inhibitor of PP2A, did not Sorcin is a 21.6-kDa calcium-binding protein that is a (23). PP2B reduced CaV1.2 current in smooth muscle member of the penta EF-hand protein family and is (24), but not in neurons (25). Phosphatases can also widely distributed among mammalian tissues (33). bind to AKAPs (15). Biochemical studies have demonstrated that sorcin binds directly to both cardiac RyRs (33) and CaV1.2 (34). Caveolin-3 and β2-adrenergic receptor Although the current-voltage relationship was virtually identical in sorcin-overexpressing TG and WT myo- Caveolae are flask-shaped invaginations of the plasma cytes, the rapid component of L-type calcium current membrane that participate in vesicular trafficking and inactivation (tau-fast) was significantly accelerated in signal transduction in various cell types (26). Caveolins sorcin-overexpressing TG myocytes compared to WT are the major structural protein of caveolar membranes, controls (35). Meyers et al. (34) reported that sorcin and Caveolin-3 is expressed predominantly in muscle interacted with the C terminus (amino acid residues (27). Recently, an immunoprecipitation analysis revealed 1622 – 1748) of rabbit CaV1.2 (1640 – 1766 in human that Caveolin-3, CaV1.2, β2-adrenergic receptor (not β1- CaV1.2). This site largely overlaps the CaM-binding adrenergic receptor), G-protein-αs, adenylyl cyclase, domain (see Fig. 1). It is not known whether there is PKA, and PP2A form a macromolecular signal complex synergistic activity between sorcin and CaM. in cardiomyocytes (28). Silencing of Caveolin-3 mRNA using siRNA eliminated the response to β2-adrenergic SNARE proteins and synaptotagmin stimulation of the L-type calcium current in neonatal mouse cardiomyocytes. This report led us to speculate SNARE (soluble N-ethylmaleimide-sensitive factor that Caveolin-3 is necessary for the regulation of the attachment protein receptor) proteins are characterized CaV1.2 channel by the β2-adrenergic receptor. However, by the presence of a SNARE motif, which is a coiled- studies in the brain, which lacks Caveolin-3 expression coil domain of 60 – 70 amino acids critical for SNARE (27), revealed regulation of the CaV1.2 channel by β2- complex formation (36). In neurons, SNARE proteins adrenergic stimulation (29). Therefore, future experi- mediate secretion of neurotransmitters from the nerve ments are necessary to reveal the detailed mechanism of terminal (37). X-ray crystallography has revealed that β2-adrenergic receptor regulation of the CaV1.2 channel. the target membrane SNARE protein (t-SNARE or Q- Molecules Interacting With Cav1.2 5

SNARE), syntaxin 1A(Sx1A) and SNAP-25, and the These reports suggest that all members of the RGK vesicle membrane SNARE protein (v-SNARE or R- family may interfere with the interaction between the β SNARE), synaptobrevin, can form a SNARE complex subunit and CaV1.2 and with the trafficking of CaV1.2 to (36). Synaptotagmin 1 (Syt1) is localized on synaptic the plasma membrane, resulting in a reduction of the vesicles and is thought to be a vesicular calcium sensor CaV1.2 current. However, surface biotinylation studies (38). Wiser et al. (39) reported that Sx1A, SNAP-25, and by Finlin et al. (46, 50) demonstrated that Rem and Syt1 can bind to the II-III linker region of CaV1.2 (amino Rem2 did not reduce the membrane expression of acid residues 753 – 893 in rat CaV1.2, 753 – 893 in CaV1.2. Co-expression of CaV1.2 and the β2 subunit with human CaV1.2) based on a GST pull-down assay. Rem did not block expression of CaV1.2 on the cell 2+ Whole-cell Ba currents mediated by CaV1.2/β2a/α2/δ surface (49). Therefore, the nature of the mechanisms subunits co-expressed with Sx1A and Syt1 in Xenopus regulating calcium channel activity by RGK family oocytes are greater than those mediated by CaV1.2 members remains unclear. In addition, Rad and Rem are /β2a/α2/δ subunits co-expressed with Sx1A alone (39, robustly expressed in the heart, but the physiological 40). These reports led us to speculate that when a role of Rad and Rem in the heart remains unclear synaptic vesicle is located near the target membrane, (45, 47). the calcium current in this area is increased due to interaction of CaV1.2 with Syt1. Subsequently, Syt1 senses AHNAK1 the increase in the calcium concentration, resulting in fusion of the synaptic vesicle with the target membrane. Ahnak (meaning “giant” in Hebrew)1 is a 700kDa, Recently, Cho et al. (41) demonstrated the tight associa- 5643aa protein also known as desmoyokin (52). tion of CaV1.2 with syntaxin 2 and SNAP-23. Vendel Hohaus et al. (53) revealed that ahnak1 is expressed in et al. (42) reported that β4 interacts with Syt1. cardiomyocytes and showed that the C terminus of ahnak1 interacts with the β2 subunit. Recently, the CSN5 Ile5236Thr mutation of Ahnak1 was identified in patients with hypertrophic cardiomyopathy. Haase et al. COP9 signalosome subunit 5 (CSN5), also known as (54) showed that the application of a C terminal ahnak1 Jun activation domain-binding protein 1 (Jab1), is fragment (amino acid residues 5215 – 5288) containing thought to be a group of coactivators that stabilizes the Ile5236Thr mutation to cardiomyocytes resulted in complexes of the transcription factors cJun or JunD, so an increase in the calcium current as well as a slight as to increase the specificity of target gene activation by leftward shift in its voltage dependence. These effects AP-1 proteins (43). Kameda et al. (44) reported that are similar to those of isoprenaline in normal cardio- CSN5 interacts specifically with the II-III linker region myocytes. The Ile5236Thr mutated ahnak1 peptide of CaV1.2 (amino acid residues 775 – 994 in rat CaV1.2, induced partial dissociation of the wild-type ahnak1- 745 – 984 in human CaV1.2). Silencing of CSN5 mRNA fragment and the β2 subunit. PKA-mediated phosphory- using siRNA decreased the endogenous protein level of lation of both ahnak1 and the β2 subunit also led to the CSN5 and activated CaV1.2 current in COS7 cells. partial dissociation of the wild-type ahnak1-fragment/β2 However, the physiological role of CSN5 in native cells subunit complex. Therefore, they proposed that β- remains unclear. adrenergic stimulation leads to partial dissociation of the ahnak1/β2 subunit complex, followed by an increase RGK family in the calcium current though CaV1.2. Stimulation of the β-adrenergic receptor stimulation in vivo by intra- Rem, Rem2, Rad, and Gem/Kir are members of a myocardial infusion of isoproterenol resulted in sub- Ras-related GTPase protein subfamily (RGK family) stantial phosphorylation of membrane-localized ahnak1, that exhibit distinct patterns of tissue-specific expression but not the β2 subunit (52). These reports led us to [Rem (45), Rem2 (46), Rad (47), and Gem/Kir (48)]. speculate that ahnak1 may be an important target of They have all been shown to interact directly with the β PKA-mediated phosphorylation in the enhancement of subunit, resulting in the down-regulation of calcium L-type calcium current by the β-adrenergic receptor. channel function [Rem (45, 49, 50), Rem2 (46), Rad (45), and Gem/Kir (48)]. The AID peptide of CaV1.2 Summary and perspectives inhibited the binding of Gem to the β3 subunit. Immuno- fluorescence microscopy showed that co-expression of In this review, we described the molecules that can CaV1.2 and the β3 subunit with Gem/Kir or Rem2 bind and regulate the CaV1.2 channel. CaM can bind to blocked cell-surface expression of CaV1.2 (48, 51). two different elements of the IQ-region of CaV1.2, 6 T Kobayashi et al indicating that binding of CaM to the IQ region may voltage-gated calcium channel regulation from the structure of produce differential effects on the kinetics of CaV1.2 the CaV1.2 IQ domain-Ca2+/calmodulin complex. Nat Struct current. This model led us to speculate that other mole- Mol Biol. 2005;12:1108–1115. 8 Zhou H, Yu K, McCoy KL, Lee A. Molecular mechanism for cules in addition to CaM may have differential effects on divergent regulation of Cav1.2 Ca2+ channels by calmodulin channel kinetics following interaction with the same and Ca2+-binding protein-1. J Biol Chem. 2005;280:29612– region of CaV1.2. 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