Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels

Abstract

Calmodulin regulation of CaV channels is a prominent Ca(2+) feedback mechanism orchestrating vital adjustments of Ca(2+) entry. The long-held structural correlation of this regulation has been Ca(2+)-bound calmodulin, complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (individually transformed Langmuir analysis). Accordingly, we uncover frank exchange of Ca(2+)-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca(2+)-calmodulin binds an N-terminal spatial Ca(2+) transforming element module on the channel amino terminus, whereas the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.

Figure 1. General schema for CaM regulation of representative L-type Ca_V1.3 channels

(a) Primary configurations of CaM/channel complex with respect to CaM-regulatory phenomena (E, A, I_C, I_N, and I_CN). Inset at far right, cartoon of main channel landmarks involved in CaM regulation, with only the pore-forming α_1D subunit of Ca_V1.3 diagrammed. Ca²⁺-inactivation (CI) region, in the proximal channel carboxy terminus (~160 aa), contains elements potentially involved in CaM regulation. IQ domain (IQ), comprising the C-terminal ~30 aa of the CI segment, long proposed as preeminent for CaM/channel binding. Dual vestigial EF-hand (EF) motifs span the proximal ~100 aa of the CI module; these have been proposed to play a transduction role in channel regulation. Proximal Ca²⁺-inactivating (PCI) region constitutes the CI element exclusive of the IQ domain. NSCaTE on channel amino terminus of Ca_V1.2–1.3 channels may be the N-lobe Ca²⁺/CaM effector site. (b) Whole-cell Ca_V1.3 currents expressed in HEK293 cell, demonstrating CDI in the presence of endogenous CaM only. CDI observed here can reflect properties of the entire system diagrammed in a, as schematized by the stick-figure diagram at the bottom of b. Here and throughout, the vertical scale bar pertains to 0.2 nA of Ca²⁺ current (black); and the Ba²⁺ current (gray) has been scaled ~3-fold downward to aid comparison of decay kinetics, here and throughout. Horizonal scale bar, 100 ms. (c) Currents during overexpression of CaM_WT, isolating the behavior of the diamond-shaped subsystem at bottom. (d) Currents during overexpression of CaM₁₂, isolating C-lobe form of CDI. (e) Currents during overexpression of CaM₃₄, isolating N-lobe form of CDI. (c–e) Vertical bar, 0.2 nA Ca²⁺ current. Timebase as in b.

Figure 2. Probing functionally relevant CaM regulatory interactions via iTL analysis

(a) Isolated C- or N-lobe regulatory system (denoted by stick-figure diagrams on left) can be coarsely represented by a five-state scheme on right. A single lobe of apoCaM begins preassociated to channel (state 1). Following disassociation (state 2), CaM may bind two Ca²⁺ ions (state 3, black dots). Ca²⁺/CaM may subsequently bind to channel effector site (state 4). From here, transduction step leads to state 5, equivalent to CDI. Association constant for lobe of apoCaM binding to preassociation site is ɛ; whereas γ₁ and γ₂ are association constants for respective transitions from states 3 to 4, and states 4 to 5. (b) Unique Langmuir relation (Eq. 1) that will emerge upon plotting channel CDI (defined Fig. 1b, right) as a function of K_a,EFF (association constant measured for isolated channel peptide), if K_a,EFF is proportional to one of the actual association constants in the scheme as in a. Black symbols, hypothetical results for various channel/peptide mutations; green symbol, hypothetical wild type. (c) Predicted outcome if peptide association constant K_a,EFF has no bearing on association constants within holochannels. (d) Outcome if mutations affect holochannel association constants, but not peptide association constants. (e) Outcome if mutations affect holochannel association constant(s) and peptide association constant, but in ways that are poorly correlated.

Figure 3. Inconsistencies with IQ domain role as Ca²⁺/CaM effector site

(a) No appreciable deficit in isolated N-lobe CDI upon point alanine substitutions across the IQ domain (sequence at top with bolded isoleucine at ‘0’ position). Left, corresponding subsystem schematic. Middle, bar-graph summary of CDI metric, as defined in Fig. 1b. Bars, mean ± SEM for ~6 cells each. Green dashed line, wild-type profile; red bar, I[0]A; blue symbol in all panels, Y[3]D. Right, exemplar currents, demonstrating no change in N-lobe CDI upon I[0]A substitution. Horizontal scale bar, 100 ms; vertical scale bar, 0.2 nA Ca²⁺ current. Red, Ca²⁺ current; gray, Ba²⁺ current. (b) Isolated C-lobe CDI (corresponding subsystem schematized on left) exhibits significant attenuation by mutations surrounding the central isoleucine (colored bars). Format as in a. I[0]A shows the strongest attenuation (red bar and exemplar currents at right). Bars average ~5 cells ± SEM . Dashed green line, wild-type profile. Timebase as in b; vertical scale bar, 0.2 nA Ca²⁺ current . (c) Bar-graph summary of association constants (K_a,EFF = 1 / K_d,EFF) for Ca²⁺/CaM binding to IQ, evaluated for constructs exhibiting significant effects in b (colored bars, with I[0]A in red), or chosen at random (hashed in b). Error bars, nonlinear standard-deviation estimates. FRET partners schematized on the left, and exemplar binding curves on the right for I[0]A (red) and wild-type (black). Symbols average ~7 cells. Smooth curve fits, 1:1 binding model. Calibration to efficiency E_A = 0.1, far right vertical scale bar. Horizontal scale bar corresponds to 100 nM. (d) Plots of N-lobe CDI versus K_a,EFF deviate from Eq. 1, much as in Fig. 2c. Green, wild type; red, I[0]A; blue, Y[3]D. (e) Plots of C-lobe CDI versus K_a,EFF also diverge from Langmuir, as in Fig. 1e. This result further argues against the IQ per se acting as an effector site for the C-lobe of Ca²⁺/CaM. Symbols as in d. (d, e) Y[3]D (blue symbol, CDI mean of 4 cells) yields poor Ca²⁺/CaM binding, but unchanged CDI. Supplementary Note 7, further FRET data.

Figure 4. iTL analysis of Ca²⁺/CaM effector role of NSCaTE module of Ca_V1.3 channels

(a) Cartoon depicting NSCaTE as putative effector interface for N-lobe of Ca²⁺/CaM. (b) Exemplar Ca_V1.3 whole-cell currents exhibiting robust isolated N-lobe CDI, as seen from the rapid decay of Ca²⁺ current (black trace). Corresponding stick-figure subsystem appears on the left. W[44]A mutation abolishes N-lobe CDI, as seen from the lack of appreciable Ca²⁺ current decay (red trace). Gray trace, averaged Ba²⁺ trace for wild-type (WT) and W[44]A constructs. Horizontal scale bar, 100 ms; vertical scale bar, 0.2 nA Ca²⁺ current (red, W44A; black, WT). (c) FRET 2-hybrid binding curves for Ca²⁺/CaM₃₄ and NSCaTE segment, with FRET partners schematized on the left. Wild-type pairing (WT) in black; W[44]A mutant pairing in red. Each symbol, mean ± SEM of ~5 cells. (d) Bar-graph summary of N-lobe CDI for NSCaTE mutations measured after 800-ms depolarization, with NSCaTE sequence at the top, as numbered by position within Ca_V1.3.. Data for W[44]A in red; dashed green line, wild type. Bars, mean ± SEM of ~5 cells. (e) Association constants (K_a,EFF = 1 / K_d,EFF) for Ca²⁺/CaM₃₄ binding to NSCaTE module evaluated for constructs exhibiting significant effects in panel d. Error bars, nonlinear standard deviation estimates. Data for W[44]A in red; dashed green line, wild type. (f) Plotting N-lobe CDI versus K_a,EFF uncovers a Langmuir, identifying NSCaTE as functionally relevant effector site. W[44]A in red; wild type in green. (g-l) iTL fails to uphold NSCaTE as effector site for C-lobe of Ca²⁺/CaM. Format as in a–f. (h, j) C-lobe CDI at 300 ms, unchanged by NSCaTE mutations. Bars in j, mean ± SEM of ~5 cells. (i, k) Changes in K_a,EFF of NSCaTE module for Ca²⁺/CaM₁₂ via 3³-FRET. Each symbol in i, mean ± SEM of ~5 cells. (l) C-lobe CDI versus K_a,EFF deviates from Langmuir, as in Fig. 2c.

Figure 5. iTL analysis of PCI segment as C-lobe Ca²⁺/CaM effector interface

(a) Channel cartoon depicting PCI segment as putative effector site for C-lobe of Ca²⁺/CaM. (b) Isolated N-lobe CDI for wild type (WT) and LGF→AAA (LGF) mutant channels. Ca²⁺ current for WT in red, and for LGF in red. Gray, averaged Ba²⁺ trace. Horizontal scale bar, 100 ms; vertical scale bar, 0.2 nA Ca²⁺ current (red, LGF; black, WT). (c) Isolated C-lobe CDI for WT and LGF mutant channels, indicating strong CDI attenuation by LGF mutation. Format as in b. (d) FRET 2-hybrid binding curves for Ca²⁺/CaM pitted against PCI segments, for WT (black) and LGF (red). Each symbol, mean ± SEM from ~ 9 cells. (e) Bar-graph summary confirming no appreciable reduction of isolated N-lobe CDI, over all alanine scanning mutants across the PCI region (sequence at the top). Schematic of corresponding system under investigation at the left. Green dashed line, wild type; red, LGF mutant; gaps, nonexpressing configurations. Bars, mean ± SEM of ~5 cells. (f) Bar-graph summary, C-lobe CDI for alanine scan of PCI. Red bar, LGF→AAA mutant showing strong CDI reduction. Rose bar, other loci showing substantial CDI reduction. Hashed, randomly chosen loci for subsequent FRET analysis below. Bars, mean ± SEM of ~5 cells. (e, f) CDI decrease for YLT cluster (Fig. 5e, f) reflects reduced Ca²⁺ entry from 30-mV depolarizing shift in activation, not CDI attenuation per se. Shifts for all other loci were at most ±10 mV (not shown). (g) Association constants for Ca²⁺/CaM binding to PCI region, with FRET partners as diagrammed on the left. Green dashed line, wild-type profile. PCI mutations yielding large C-lobe CDI deficits were chosen for FRET analysis (red and rose in f), as well as those chosen at random (hashed in f). Error bars, nonlinear estimates of standard deviation. (h) Plots of N-lobe CDI versus K_a,EFF for Ca²⁺/CaM binding to PCI deviated from Langmuir. Red, LGF; green, WT. (i) Alternatively, plotting C-lobe CDI revealed Langmuir relation, supporting PCI as C-lobe Ca²⁺/CaM effector site. Symbols as in h.

Figure 6. Role of IQ domain in C-lobe CDI

(a) Cartoon depicting putative binding interaction between IQ domain and PCI segment, which is also required for C-lobe CDI. (b) Bar-graph summary of C-lobe CDI measured for alanine scan of IQ domain, reproduced from Fig. 3b. Strongest CDI reduction for I[0]A mutant (red), followed closely by loci affiliated with rose and blue bars underneath gray dashed-line. Dashed-green line, wild-type. (c) Association constants K_a,EFF determined for 3³-FRET binding between IQ domain and PCI region (partners diagrammed at left), under elevated levels of Ca²⁺. Wild-type profile, green dashed line. Bars, K_a,EFF for mutants with strongest effects (colored bars in b) or chosen at random (hashed bars in b). Error bars, nonlinear standard deviation estimates. (d) Exemplar 3³-FRET binding curves for IQ/PCI interaction. Each symbol, mean ± SEM of ~8 cells. Absent Ca²⁺, the IQ domain associates only weakly with the PCI region (gray). However, elevated Ca²⁺ greatly enhances binding (black). (e) 3³-FRET binding curves for I[0]A (red) and Q[1]A (blue) mutations under elevated Ca²⁺. Each symbol, mean ± SEM of ~5 cells. Fit for wild type IQ/PCI interaction reproduced from d in black. (f) Plotting C-lobe CDI versus K_a,EFF under elevated Ca²⁺ unveils a well-resolved Langmuir relation. WT (green), I[0]A (red), and Q[1]A (blue).

Figure 7. Footprint of apoCaM preassociation with the PCI segment

(a) Channel cartoon depicting apoCaM preassociated with the CI region, with C-lobe engaging IQ domain, and N-lobe associated with PCI region. (b) Whole-cell currents for TVM→AAA mutant in the PCI segment (Ca²⁺ in red; Ba²⁺ in gray), with only endogenous CaM present. Horizontal scale bar, 100 ms; vertical scale bar, 0.2 nA Ca²⁺ current. (c) Overexpressing CaM_WT rescues CDI for TVM→AAA mutation, suggesting that PCI region harbors an apoCaM preassociation locus. Format as in b. (d) 3³-FRET binding curves show strong apoCaM binding to CI region. Wild type (WT) in black; TVM→AAA in red. Each symbol, mean ± SEM of ~7 cells. (e) Bar-graph summary of CDI with only endogenous CaM present (CDI_CaMendo), across alanine scan of PCI region. TVM→AAA (red) shows strongest effect, with rose colored bars also showing appreciable CDI reduction. Bars, mean ± SEM of ~5 cells. Left, schematic of relevant CaM subsystem. (f) Bar-graph summary of CDI rescue upon overexpressing CaM_WT (CDI_CaMhi), for mutations showing significant loss of CDI (colored bars in e), or chosen at random (hashed bars in e). Bars, mean ± SEM of ~5 cells. Corresponding subsystem of regulation on the left. (g) Bar-graph summary of K_a,EFF for apoCaM binding to CI region, with partners as sketched on the left. Data obtained for nearly all mutants with significant CDI reduction (colored in e), and for some mutants chosen at random (hashed in e). Error bars, nonlinear estimates of standard deviation. (h) iTL analysis confirms role of PCI as functionally relevant apoCaM site. Plotting CDI_CaMendo/CDI_CaMhi (e and f) versus K_a,EFF for apoCaM/CI binding uncovers well-resolved Langmuir relation. TVM→AAA, red; WT, green. (i) Overlaying like data for IQ-domain analysis presented elsewhere (blue symbols here) displays remarkable agreement, consistent with the same apoCaM binding both PCI and IQ domains. Deep blue symbols, A[−4], I[0], F[+4] hotspots for apoCaM interaction with IQ element.

Figure 8. New view of CaM regulatory configurations of Ca_V1.3 channels

(a) Molecular layout of configurations A, I_C, I_N, and I_CN for conceptual scheme in Fig. 1a. ApoCaM preassociates with CI region: C-lobe articulates IQ domain, and N-lobe engages the PCI segment. Once Ca²⁺ binds CaM, the N-lobe of Ca²⁺/CaM departs to NSCaTE on channel amino terminus, eliciting N-lobe CDI (I_N). Alternatively, the C-lobe of Ca²⁺/CaM migrates to PCI segment, recruiting IQ domain to tri-partite complex (I_C). Finally, I_CN corresponds to channel that has undergone both N- and C-lobe CDI. (b) De novo model of Ca_V1.3 CI region docked to apoCaM (PCI region: green; IQ domain: blue). ApoCaM hotspots (Fig. 6e–g) in red. C-lobe of apoCaM contacts IQ, while N-lobe binds EF-hand region. (c) Left, atomic structure of NSCaTE bound to N-lobe of Ca²⁺/CaM (2LQC). NSCaTE peptide in tan; and N-lobe Ca²⁺/CaM in cyan. Ca²⁺, yellow. N-lobe CDI hotspots on NSCaTE in red. Right, de novo model of tripartite IQ-PCI-Ca²⁺/CaM complex (PCI region, green; IQ domain, blue). C-lobe CDI hotspots in red for both PCI and IQ domains.