Calmodulin overexpression does not alter Ca_v1.2 function or oligomerization state

Abstract

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca_vs) are crucial for Ca_v activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca²⁺/CaM molecules bound to the Ca_v1.2 C-terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca²⁺/CaM bridged two PreIQ helixes to form a 4:2 Ca²⁺/CaM:Ca_v C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM overexpression has no functional effect on Ca_v1.2 inactivation or on the stoichiometry of full-length Ca_v1.2. These data provide further support for the monomeric Ca_v1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca²⁺/CaM:Ca_v C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.

Introduction

High-voltage activated voltage-gated calcium channels (Ca_v1s and Ca _v2s) couple membrane depolarization dependent calcium entry to many physiological processes.^1,2 These channels are multi-subunit complexes that include a Ca_v α₁ pore-forming subunit, the auxiliary proteins Ca_vβ, Ca_vα₂δ and calmodulin (CaM).^1,3,4 Calcium-calmodulin (Ca²⁺/CaM) has a central role in two distinct Ca_v feedback mechanisms, calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF),⁵ that involve direct interactions with the Ca_vα₁ C-terminal cytoplasmic tail IQ domain.^6–12 Understanding the molecular basis for CaM-Ca_v interactions and how these interactions affect channel function remains an important challenge that requires structural definition and biochemical analysis of key regulatory CaM binding sites.

Results and Discussion

We recently reported the structure of a complex of Ca²⁺/CaM and a segment of the Ca_v1.2 C-terminal tail that encompasses the PreIQ and IQ domains (residues 1,561–1,637).⁸ Ca²⁺/CaM has two lobes, N-lobe and C-lobe, which can each engage target-binding partners. Consequently, a number of diverse Ca²⁺/CaM-target binding modes have been reported.^13,14 The Ca²⁺/CaM:Ca_v1.2 C-terminal tail complex defines two Ca²⁺/CaM binding sites that are occupied by two separate Ca²⁺/CaMs. Each interacts with the channel in distinct ways (Fig. 1A). One Ca²⁺/CaM engages the IQ domain using both lobes in a manner identical to prior structures of Ca²⁺/CaM-Ca_v1.2 IQ domain complex.^12,15 In contrast, the second Ca²⁺/CaM binds the C-terminal segment of the long PreIQ helix using only Ca²⁺/C-lobe. Notably, the association of CaM with the PreIQ-IQ segment creates a number of electronegative patches (Fig. 1B and C). These patches raise the possibility that positively charged residues elsewhere in the channel form key interactions with this segment.

Figure 1.

Structure of the 2:1 Ca²⁺/CaM:Ca_v1.2 C-terminal tail complex. (A) Cartoon diagram of the Ca²⁺/CaM:Ca_v1.2 C-terminal tail complex. The Ca_v1.2 C-terminal tail is shown in red. Ca²⁺/CaM molecules bound to the PreIQ and domains are indicated. In both cases Ca²⁺/CaM is shown using a surface representation and the Ca²⁺/N-lobes and Ca²⁺/C-lobes of the PreIQ bound and IQ bound Ca²⁺/CaMs are colored forest and dark blue, and green and marine, respectively. (B) Cartoon diagram as in ‘(A)’ but lacking the Ca²⁺/N-lobe from the PreIQ Ca²⁺/CaM. Colors as in (A). (C) Surface charge representation of the 2:1 Ca²⁺/CaM:Ca_v C-terminal tail shown in the same orientation as ‘(B)’. Colors indicate a scale from −10 kT/e (red) to + 10kT/e (blue).

Biochemical studies revealed that the two Ca²⁺/CaMs have very different properties. The Ca²⁺/CaM molecule bound to the PreIQ segment is labile and could be removed by passage over a phenylsepharose column in conditions of high-calcium (2 mM). In contrast, the Ca²⁺/CaM on the IQ domain remained bound. Further, functional studies indicated that the PreIQ site has a role in calcium-dependent facilitation (CDF).⁸ Extensive biophysical characterization showed that the complex maintains a 2:1 Ca²⁺/CaM:C-terminal tail stoichiometry even at protein concentrations that match those found in the most densely packed native Ca_v environment (≥100 µM). Together, these studies demonstrate that the Ca_v1.2 C-terminal tail can bind two Ca²⁺/CaMs simultaneously and suggest that the Ca²⁺/CaM occupying the PreIQ binding site is poised to make interactions with other elements of the channel by virtue of the free Ca²⁺/N-lobe.

A curious feature of the asymmetric unit of the Ca²⁺/CaM:PreIQ-IQ complex was that it contained two Ca²⁺/CaM:PreIQ-IQ complexes that interacted through a portion of the PreIQ domain and was bridged by the PreIQ-Ca²⁺/CaMs. A similar X-ray structure led to the proposal that Ca_v1.2 channels dimerize via this sort of interaction and that this interaction is functionally relevant.¹⁶ It is not straightforward to discern the relevance of protein-protein interactions from crystal lattice contacts alone as such interactions are a necessary requirement for forming a crystal lattice.^17,18 Further, there are numerous examples in which crystallographic dimers do not represent biologically relevant interactions.¹⁹

Our biophysical data on the isolated Ca_v1.2 channel tail complex clearly indicated a 2:1 stoichiometry. Although it has not been generally thought that Ca_v channels function as dimers, electron microscopy studies of full-length Ca_v1.2 ^20,21 and the observation of infrequent multiple concerted Ca_v1.2 openings²² have suggested that under some conditions the full-length channel may form higher order assemblies. To address the question of Ca_v1.2 oligomerization state in live cell membranes directly, we used total internal reflection fluorescence microscopy (TIRFM) ²³ and Ca_v1.2 bearing a C-terminal green fluorescent protein (GFP) tag for single-molecule subunit counting. Our studies showed unambiguously that the Ca_v1.2 channels are monomeric.⁸ Importantly, the Navedo et al. study showed that deletion of a large section of the C-terminal tail (D1670X), which removes all of the C-terminal tail after the IQ domain, eliminates all measurable multichannel coupling. Thus, unlike what has been proposed by Fallon et al. the Ca_v1.2 4:2 complex observed in the crystal structure is non-physiological and is not relevant for channel function. Further, whatever coupling mechanism underlies the observations of Navedo et al. it does not involve the PreIQ-IQ domain.

A key assumption of both our functional and subunit counting experiments is that the Xenopus oocytes that are used to overexpress Ca_v1.2 have sufficient endogenous CaM to make functional channels. Previous studies have shown that multiple alanine mutations on the channel that weaken CaM affinity for the IQ domain and reduce CDI can be overcome, at least in part, by CaM co-expression.^24,25 Because the PreIQ-bound CaM is responsible for crossbridging interactions in the asymmetric unit and is labile, we tested whether CaM overexpression would affect Ca_v1.2 function or oligomerization state. Injection of Xenopus oocytes with fixed amounts of Ca_v1.2 mRNA and either a 10-fold or 60-fold excess of CaM mRNA gave Ca_v1.2 channels in which both CDI and a second form of inactivation, voltage-dependent inactivation (VDI), were indistinguishable from Ca_v1.2 relying on endogenous CaM (Fig. 2A and Table 1).

Figure 2.

Effect of CaM overexpression on Ca_v1.2 function and stoichiometry. (A) Comparison of Ca_v1.2 CDI and VDI as a function of CaM overexpression. Normalized Ca²⁺ and Ba²⁺ currents for channels relying on endogenous CaM (black and gray, respectively) or with excess CaM mRN A at the indicated ratios (dark blue and light blue, respectively). (B) Normalized Ca²⁺ and Ba²⁺ currents recorded from Ca_v1.2-GFP co-expressed with CaM? mCherry at a mRNA ratio of 1:10 (dark blue and light blue, respectively) are compared to wild type (black and gray, respectively). Experimental details are the same as in reference ⁸. (C) Western blot detection of whole-cell lyates and immunoprecipitation of Flag-tagged Ca_v1.2 from Xenopus oocytes. Detection was with an antibody against Flag or mCherry as indicated. (D) Subunit counting of Ca_v1.2 in live cell membranes. A representative TIRF image showing the Ca_v1.2-GFP fluorescent spots at the cell surface of X. laevis oocytes. Bright spots are Ca_v1.2-GFP single-molecules when the shutter is first opened at the beginning of the bleaching experiment. Blue circles mark the selected molecules for subunit counting. (E) TIRF image showing CaM-mCherry spots in the cell surface of X. laevis oocytes. (F) Time courses of photobleaching from single GFP fluorescent spots. Two examples are shown. (G) Distribution of fluorescent spots that bleach in one or more steps. Percentile of each bleaching step is indicated.

Table 1.

Ca_v1.2 inactivation parameters

	ti300 (%)	A1 (%)	τ1 (ms-1)	A2 (%)	τ2 (ms-1)	N
Cav1.2	69.0 ± 2.2	55.8 ± 3.2	25.0 ± 3.7	16.4 ± 1.9	138 ± 21	12
Cav1.2, CaM 1:10	67.7 ± 3.5	53.9 ± 7.3	26.7 ± 4.2	17.5 ± 5.9	136 ± 32	8
Cav1.2, CaM 1:60	69.0 ± 2.4	57.2 ± 1.7	25.0 ± 4.8	15.9 ± 3.3	142 ± 37	6
Cav1.2-GFP, CaM-Cherry 1:10	71.4 ± 7.3	55.2 ± 6.5	25.3 ± 5.3	20.5 ± 4.6	142 ± 29	8

All experiments correspond to the mean of at least two separate oocyte batches. ‘±’ values are standard deviation.

To examine whether the overexpressed CaM associated with Ca_v1.2, we coinjected mRNA for Flag-tagged Ca_v1.2 and mCherry-labeled CaM at a ratio of 1:10. This combination has identical functional properties to the untagged components (Fig. 2B and Table 1). Both components are clearly overexpressed (Fig. 2C). Immunoprecipitation using anti-Flag M2 agarose to capture the Ca_v1.2 subunit did not detect any interaction between Ca_v1.2 and CaM in the absence of Ca²⁺ (Fig. 2C). Importantly, similar to prior reports with untagged subunits,²⁶ in the presence 500 µM Ca²⁺ the CaM-mCherry-Ca_v1.2 interaction was readily observed and directly demonstrated that overexpressed CaM binds the channel (Fig. 2C). These data, together with the absence of functional effects of CaM overexpression, strongly suggest that the endogenous levels of CaM present in Xenopus oocytes are sufficient to account for all Ca_v1.2 functional effects.

We also examined by single molecule subunit counting whether excess CaM expression influenced Ca_v1.2 oligomerization (Fig. 2D–G). TIRF images indicated that CaM-mCherry was present at the membrane. Examination of spots in which both Ca_v1.2-GFP and CaM-mCherry were present showed that the vast majority (95%) of Ca_v1.2-GFP fluorescent spots bleached in a single step (Fig. 2F and G). These data provide strong indication that the channels remain monomers even when excess CaM is present at the membrane and further support our interpretation that the structure of CaM bridging two PreIQ helices is not relevant for full-length channels.

Our studies demonstrate that the Ca_v1.2 PreIQ-IQ segment is capable of binding multiple Ca²⁺/CaMs simultaneously and that the PreIQ site is involved in channel function.⁸ The organization of multiple Ca²⁺/CaMs along a largely helical segment is reminiscent of how CaM and CaM-like proteins are organized on myosin²⁷ and raises the intriguing possibility that there are previously unrecognized connections between these systems. How the Ca²⁺/CaM:Ca_v1.2 C-terminal tail complex interacts with other channel elements in both the calcium-bound and calcium-free states remains a major open question. The presence of large patches of electronegative surface on the Ca²⁺/CaM:Ca_v1.2 C-terminal tail complex raises the possibility that there may be positively charged residues elsewhere in the channel that form key interactions with this segment. Defining such interactions and their state-dependence remains an important goal for the field.

Methods

Two-electrode voltage clamp recordings.

Homo sapiens CaM (GenBank NM_006888) was subcloned into pGEMHE-mCherry plasmid²⁸ using SacII/HindIII restriction sites. All other DNA constructs were the same as in reference ⁸. Generation of mRNA and electrophysiological protocols were as described previously in references 8 and 12, except where noted. In brief, Xenopus oocytes were injected with 50 nl RNA mixture of Ca_v1.2 (or Ca_v1.2-mEGFP), Ca_vβ_2a, Ca_vα₂δ-1 and CaM (or CaM-mCherry) at a molar ratio of 1:1:1:10 unless indicated otherwise and recorded 2–4 days after injection. All data were produced from more than one oocyte batch and analyzed with Clampfit 9.2 (Axon Instruments).

Western blot detection of whole-cell lyates and immunoprecipitation of flag-tagged Ca_v1.2 from Xenopus oocytes.

In each case, nineteen oocytes were injected with Ca_v1.2-3xFlag, Ca_vβ_2a, Ca_vα₂δ and CaM-mCherry at a molar ratio of 1:1:1:10 or water. After two days, oocytes were collected and homogenized in immunoprecipitation (IP) buffer 150 mM NaCl, 0.5% CHAPS, 50 mM Hepes/NaOH pH 7.4 and Complete protease inhibitors (Roche), with or without 500 µM CaCl₂ (IP + Ca²⁺ or IP-Ca²⁺ respectively). Lysates were cleared by centrifugation at 13,000 rpm for 30 min and 600 µg of protein was incubated with 35 µl anti-Flag M2 agarose beads (Sigma-Aldrich) overnight at 4°C. Beads were washed four times with IP buffer and bound proteins were released by heating at 70°C for 10 min in NuPAGE sample buffer (Invitrogen). Samples were separated on a 4–12% NuPAGE Bis-Tris gel, transferred on nitrocellulose and reacted with either anti-FLAG monoclonal antibody (1:5,000, Sigma-Aldrich) or Living Colors DsRed polyclonal antibody (1:1,000, Clontech).

Subunit counting experiments.

DNA constructs for Ca_v1.2-mEGFP and CaM-mCherry were the same as described above and in reference ⁸. RNA transcription and experimental details are as described in reference ⁸, except where noted below. Oocytes were injected with 50 nl of a 1:1 mixture of 500 ng/µl Ca_v1.2-EGFP and 100 ng/µl Ca_vβ_2a cRNA and 150 ng/µl CaM-mCherry cRNA.

Addendum to: Kim EY, Rumpf CH, Van Petegem F, Arant RJ, Findeisen F, Cooley ES, et al. Multiple C-terminal tail Ca2+/CaMs regulate Cav1.2 function but do not mediate channel dimerization. EMBO J. 2010;29:3924–3938. doi: 10.1038/emboj.2010.260.