Pore mutation N617D in the skeletal muscle DHPR blocks Ca2+ influx due to atypical high-affinity Ca2+ binding

Abstract

Skeletal muscle excitation-contraction (EC) coupling roots in Ca²⁺-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca²⁺ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca²⁺ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca²⁺-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca²⁺ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca²⁺ pore-binding is tight for physiologically relevant monovalent cations like Na⁺ and K⁺. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

Keywords: ca2+ permeation; ca2+ selectivity; molecular biophysics; mouse; nc dhpr; non-conducting dhpr; pore binding affinity; skeletal muscle excitation-contraction coupling; structural biology; voltage-gated ca2+ channel.

Figure 1.. Mutant DHPR(N617D) remains Ca²⁺ impermeant despite strong or long depolarizations in the presence of DHP agonist Bay K.

(a) Representative whole-cell Ca²⁺ current recordings elicited by 200 ms depolarizations from −50 to +80 mV from ncDHPR (top) and wt (center) myotubes before (left) and after (right) perfusion with 10 µM of the DHP agonist (±)Bay K 8644. Scale bars, 50 ms (horizontal), 4 pA/pF (vertical). Plots of current-voltage relationship (bottom) indicates lack of DHPR inward Ca²⁺ currents in the absence (I_max = −0.02 ± 0.01 pA/pF; n = 8) and presence (I_max = −0.02 ± 0.01 pA/pF; n = 5) of (±)Bay K through ncDHPR myotubes, in contrast to significant (p<0.001) augmentation of Ca²⁺ currents in wt myotubes upon administration of (±)Bay K (No Bay K: I_max = −5.04 ± 0.27 pA/pF; n = 9; with Bay K: I_max = −8.82 ± 0.56 pA/pF; n = 6). (b) 200 ms strong depolarization to +90 mV followed by 100 ms to +60 mV and finally repolarization to −20 mV for 70 ms (bottom, green lines) in the presence of 10 µM (±)Bay K, were unable to evoke inward Ca²⁺ currents through DHPR(N617D) (top, with +90 mV prepulse: I_max = −0.02 ± 0.02 pA/pF; without +90 mV prepulse: I_max = 0.01 ± 0.02 pA/pF; n = 10). Contrary, wt DHPR displayed significant (p<0.01) depolarization-induced potentiation of inward current at +60 mV (with +90 mV prepulse: I_max = −2.97 ± 0.54 pA/pF; without +90 mV prepulse: I_max = −1.62 ± 0.37 pA/pF; n = 5) (center). Upon subsequent repolarization from +60 mV to −20 mV, the tail current was also considerably larger (p<0.01) after the +90 mV pre-conditioning pulse (I_tail = −19.36 ± 3.59 pA/pF; n = 5) (center, green trace) than after the +60 mV pulse (I_tail = −10.78 ± 1.99 pA/pF; n = 5) (center, black trace). Statistical significance was calculated using paired t-test. Scale bars, 100 ms (horizontal), 3 pA/pF (vertical). (c) Likewise, 2 s long depolarizations from +10 mV to +80 mV in 10 mV increments (bottom) in the presence of 10 µM (±)Bay K, were unable to induce Ca²⁺ influx through DHPR(N617D) (top, I_max = −0.05 ± 0.02 pA/pF; n = 5). The same voltage protocol evoked robust inward Ca²⁺ currents through wt DHPR (center, I_max = −7.69 ± 0.56 pA/pF; n = 5). Scale bars, 500 ms (horizontal), 4 pA/pF (vertical). Data are presented as mean ± SEM; p determined by unpaired Student’s t-test.

Figure 2.. Mutant DHPR(N617D) does not conduct inward Na⁺ currents in the presence of near physiological [Na⁺].

(a) Plots of current-voltage relationship for DHPR-mediated Na⁺ currents recorded from ncDHPR myotubes indicate the absence of slow-activating, non-inactivating inward Na⁺ currents in the presence of near physiological 150 mM external Na⁺ with either 10 mM (n = 8) or 1 mM external Ca²⁺ (n = 9). Control recordings were performed in standard bath solution (150 mM TEA⁺, 10 mM Ca²⁺) (n = 8). (b) Representative current recordings from ncDHPR myotubes in response to 200 ms depolarizing test pulses between −50 to +80 mV in the presence of 10 mM Ca²⁺ with either 150 mM Na⁺ (top) or 150 mM TEA⁺ (bottom), or 1 mM Ca²⁺ with 150 mM Na⁺ (center) in the bath solution. Scale bars, 50 ms (horizontal), 3 pA/pF (vertical). (c) Plots of current-voltage relationship for ncDHPR myotubes at 150 mM external Na⁺ and 1 mM external Ca²⁺ indicate no difference (p>0.05) in outward and inward currents in the presence (n = 10) and absence (n = 6) of 10 µM of the 1,4-DHP antagonist nifedipine.

Figure 3.. Binding of Ca²⁺ ions with nanomolar affinity within the pore of mutant DHPR(N617D) precludes Ca²⁺ permeation.

Representative whole-cell Li⁺ current recordings from wt and ncDHPR myotubes in response to 200 ms depolarizations from −50 to +40 mV in the presence of 100 mM external Li⁺ and either 0 (a), 1 µM (b) or 3 µM (c) free external Ca²⁺. Scale bars, 50 ms (horizontal), 1 pA/pF (vertical). Plots of current-voltage relationship are depicted at the bottom of the corresponding representative Li⁺ current traces. Inward Li⁺ currents with no blocking ion (free [Ca²⁺]=0) were indistinguishable (p>0.05) between ncDHPR (I_max = −2.32 ± 0.35 pA/pF; n = 16) and wt (I_max = −2.07 ± 0.47 pA/pF; n = 9) myotubes (a, bottom). However, at higher external [Ca²⁺] of 1 µM (b) and 3 µM (c), inward Li⁺ currents were significantly (p<0.001) smaller in ncDHPR (I_max = −0.47 ± 0.10 pA/pF, n = 8; I_max = −0.06 ± 0.02 pA/pF; n = 7, respectively) compared to wt myotubes (I_max = −1.68 ± 0.25 pA/pF, n = 6; I_max = −0.24 ± 0.04 pA/pF; n = 6, respectively). (d) Four-parameter fitted concentration-response curves of Ca²⁺ block of inward Li⁺ currents for wt and mutant ncDHPR. Averaged I/I₀ peak currents are plotted as a function of free external Ca²⁺ concentrations (up to 30 µM) and each data point is an average of 5–16 myotubes (Table 1). There is a significant (p<0.01) shift in IC₅₀ (grey dotted lines) between wt (IC₅₀ = 1.57 µM) and ncDHPR (IC₅₀ = 0.37 µM) indicating a 4.2-fold higher Ca²⁺ pore-binding affinity in the mutant DHPR(N617D) channel. Data are presented as mean ± SEM; p determined by unpaired Student’s t-test.

Figure 4.. Inward Li⁺ currents conducted by DHPR(N617D) are sensitive to nifedipine block.

(a) Plots of current-voltage relationship for DHPR-mediated Li⁺ currents recorded from ncDHPR myotubes in the presence (I_max = −0.35 ± 0.13 pA/pF; n = 16) and absence (I_max = −2.41 ± 0.27 pA/pF; n = 11) of 10 µM of the 1,4-DHP antagonist nifedipine, 100 mM external Li⁺, and free external Ca²⁺ = [0]. Maximum inward Li⁺ currents were significantly (p<0.001) reduced in the presence of nifedipine. (b) Representative whole-cell Li⁺ current recordings from ncDHPR myotubes in response to 200 ms depolarizations from −50 to +40 mV in the presence (upper) and absence (lower) of 10 µM nifedipine with 100 mM external Li⁺ and 0 external Ca²⁺ concentration. Scale bars, 50 ms (horizontal), 2 pA/pF (vertical). Data are presented as mean ± SEM; p determined by unpaired Student’s t-test.

Figure 5.. Ca²⁺ selectivity and conductance mechanisms in the wt and mutant DHPR(N617D) channel pore.

(a, b) De novo conformation prediction of peptide F₆₀₀ - I₆₂₄ constituting the selectivity filter and adjacent pore helices P1 and P2 of DHPRα_1S repeat II (P1_II, P2_II) (left) and of peptide F₁₃₀₉ - S₁₃₃₃ forming the opposite repeat IV (P1_IV, P2_IV) (right), using the program PEP-FOLD 3.5 (Thévenet et al., 2012) on the RPBS web portal. Resulting clusters from 200 independent simulations were sorted by sOPEP energy (Wang et al., 2011) to yield the ‘best model’ prediction. Biasing the model prediction of these peptides by imposing the reference structure of DHPRα_1S according to the Protein Data Bank (PDB accession number 5GJV) (Wu et al., 2016) did not lead to major differences compared to unbiased modeling approaches and hence we used unbiased models for the wt (a) and DHPR(N617D) (b) inner channel pore. Depicted best models are graphical overlays of cartoon and balls and sticks input style options. Models depict the hypothetical mechanism of Ca²⁺ conductance through the wt DHPR (a) and the block of Ca²⁺ conductance due to atypical high Ca²⁺ binding affinity (because of introduction of the negative charge D₆₁₇; boxed in red) in the DHPR(N617D) pore region. Dotted lines indicate binding interactions between Ca²⁺ ions (blue spheres) and carboxyl oxygens (red balls) of glutamate E₂₉₂ and aspartate D₂₉₆ of repeat I, E₆₁₄, D₆₁₅, and D₆₁₇ of repeat II, E₁₀₁₄ of repeat III, as well as E₁₃₂₃ and E₁₃₂₇ of repeat IV. Low affinity Ca²⁺ binding is indicated with a light blue sphere and high-affinity binding with dark blue spheres. DCS locus is the divalent cation selectivity filter (Cens et al., 2007) and EEEE locus is the Ca²⁺ selectivity filter. Vertical blue arrows indicate active Ca²⁺ conductance pathway in wt DHPR (a) and red T-bar indicates block of Ca²⁺ flux by aberrant high-affinity binding to the DCS locus in the mutant DHPR(N617D) channel pore (b). See Figure 5—figure supplement 1 for additional blocking strategies of DHPR Ca²⁺ conductance in the evolution of skeletal muscle EC coupling.

Figure 5—figure supplement 1.. Additional blocking strategies of DHPR Ca²⁺conductance in the evolution of skeletal muscle EC coupling.

Symbols and nomenclature are identical to Figure 5. (a) Zebrafish slow-muscle specific DHPRα_1S carries a distorted EEEE locus, due to substitution of E₂₉₂ of repeat I by Q. Exchange of E₂₉₂ with Q₂₉₂ (boxed in blue) in rabbit DHPRα_1S yielded mutant DHPR(E292Q) which lacks Ca²⁺ conductance (red T-bar) due to alteration of the high-affinity EEEE locus to a low affinity QEEE motif. (b) In phylogenetically advanced teleost species, the DCS locus is distorted due to charge conversion, that is exchange of D with K in repeat I. Substitution of D₂₉₆ with K₂₉₆ (boxed in blue) in rabbit DHPRα_1S yielded mutant DHPR(D296K), which lacks Ca²⁺ conductance (red T-bar) due to non-binding of Ca²⁺ to the DCS locus and thus, lack of the attraction mechanism for t-tubular Ca²⁺.

Figure 6.. Structure models of selectivity filter regions of wt DHPR (left panels) and mutant DHPR(N617D) channel pores (right panels) showing the movements of Ca²⁺ ions in simulation studies.

(a) Top view of the pore illustrating the EEEE and DCS loci. The residues of the EEEE locus are displayed in red and the DCS locus is indicated by the position of the residues N₆₁₇ or D₆₁₇. (b) Side view of wt DHPR and mutant DHPR(N617D) pores with Ca²⁺ ions present in the pore before starting the equilibration. The dark blue spheres represent van der Waals radii of the Ca²⁺ ions. (c) Snap-shots immediately following the equilibration run show that Ca²⁺ ions already moved towards the DCS and EEEE loci. While the front Ca²⁺ ion already leaves the selectivity filter of the wt DHPR toward the cytosolic side, Ca²⁺ ions in the DHPR(N617D) pore are still bound to the DCS and EEEE loci. (d) Free energy estimations from metadynamics simulations capturing the movements of Ca²⁺ ions through the selectivity filter region. The free energy profile for the passage of Ca²⁺ ions through wt DHPR selectivity filter is depicted in blue and for mutant DHPR(N617D) in red. The energy barrier of the Ca²⁺ ion leaving the wt DHPR selectivity filter (15 ± 4 kcal/mol; n = 5) is significantly smaller (p<0.001) compared to DHPR(N617D) (122 ± 20 kcal/mol; n = 5). The process was described by a one-dimensional collective variable that is, the displacement of a Ca²⁺ ion along the axis of the channel pore. A second Ca²⁺ was directly present in the simulation domain. Thus, the energy profile corresponds to the energy experienced by the first Ca²⁺ ion in the presence of the second one. See Figure 6—video 1 and Figure 6—video 2 for illustration of the movement of Ca²⁺ ions through the selectivity filter region of wt DHPR and DHPR(N617D) channel pores, respectively.