To investigate whether DHPR pore mutation N617D obstructs Ca²⁺ permeation also under current enhancing conditions, we implemented corresponding experimental protocols and measured whole-cell Ca²⁺ currents from wt and ncDHPR myotubes isolated from new born up to 4-day-old mouse pups. As a first step, inward Ca²⁺ currents were recorded in the presence of 10 µM 1,4-dihydropyridine (DHP) agonist (±)Bay K 8644 applied via the standard bath solution (see Material and methods). For voltage-gated L-type Ca²⁺ channels (Ca_V), Bay K 8644 acts as a channel opener by occupying a fenestration site at the interface of repeats III and IV in the pore region (Grabner et al., 1996; Zhao et al., 2019). Although the standard depolarization protocol (−50 to +80 mV) elicited the expected robust (±)Bay K-induced amplification (p<0.001) of Ca²⁺ currents (No Bay K: I_max = −5.04 ± 0.27 pA/pF; n = 9 and with Bay K: I_max = −8.82 ± 0.56 pA/pF; n = 6) through the wt DHPR (Figure 1a, center and bottom), no inward Ca²⁺ currents (p<0.001) (I_max = −0.02 ± 0.01 pA/pF; n = 5) or tail currents were evoked in ncDHPR myotubes under (±)Bay K 8644 administration (Figure 1a, top and bottom).

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L-type Ca²⁺ channels show a shift in the mode of gating not only by DHP agonist action (Hess et al., 1984) but also in response to strong or prolonged membrane depolarizations. As previously demonstrated (Wilkens et al., 2001), potentiation of L-type Ca²⁺ channels by DHP agonist Bay K 8644 and strong depolarizations occurs via distinct mechanisms. The shift in mode of gating, also referred to as ‘mode 2’ gating is characterized at the single-channel level by high open probability (P_O) and long mean open times (Pietrobon and Hess, 1990). Depolarization-induced entry into mode two is reflected by increased Ca²⁺ currents as well as tail currents with slower rate of current decay. To investigate whether strong depolarizations with simultaneous administration of (±)Bay K 8644 enable the entry of mutant DHPR(N617D) into mode two and elicit L-type Ca²⁺ currents, we used the pulse protocol depicted in Figure 1b (bottom) (Bannister and Beam, 2011; Bannister and Beam, 2013). Briefly, 200 ms strong, conditioning depolarization pulses from −50 mV to +90 mV, followed by a pulse of +60 mV to putatively elicit enhanced inward Ca²⁺ currents and subsequently a repolarization pulse to −20 mV to trigger tail currents were applied. As expected from wt myotubes, we recorded significantly larger inward Ca²⁺ current at +60 mV (I_max = −2.97 ± 0.54 pA/pF; n = 5; p<0.01) as well as tail current at −20 mV (I_tail = −19.36 ± 3.59 pA/pF; n = 5; p<0.01) when preceded by a pulse of +90 mV compared to the corresponding currents recorded without the pre-conditioning depolarization of +90 mV (I_max = −1.62 ± 0.37 pA/pF; I_tail = −10.78 ± 1.99 pA/pF; n = 5) (Figure 1b, center). Conversely, no inward currents or tail currents could be evoked in ncDHPR myotubes with (I_max = −0.02 ± 0.02 pA/pF; n = 10) or without the +90 mV pre-conditioning pulse (I_max = 0.01 ± 0.02 pA/pF; n = 10) (Figure 1b, top). The slight outward component at +90 mV is typically observed at strong depolarizing potentials as described previously (Schredelseker et al., 2010; Dayal et al., 2017).

Finally, beside strong depolarizations, long depolarizations are known to drive L-type Ca²⁺ channels into mode two state (Pietrobon and Hess, 1990; Bannister and Beam, 2013). Although, the 2 s depolarizations between +10 mV and +80 mV (Figure 1c, bottom) in the presence of (±)Bay K 8644 elicited robust, slowly inactivating L-type Ca²⁺ currents in wt control myotubes (I_max = −7.69 ± 0.56 pA/pF; n = 5) (Figure 1c, center), no inward Ca²⁺ currents were evoked in ncDHPR myotubes (I_max = −0.05 ± 0.02 pA/pF; n = 5) (Figure 1c, top), suggesting that DHPR(N617D) remained Ca²⁺ impermeant even under potentiating conditions. We found slight outward currents that were similar to previously observed currents in DHPRα_1S-null (dysgenic) myotubes (Bannister and Beam, 2013) recorded under identical conditions, and thus are unrelated to the DHPR.

Altogether, our results demonstrate that recording conditions known to potentiate L-type inward Ca²⁺ currents through the wt DHPR were unable to evoke Ca²⁺ currents through the mutant DHPR(N617D) in the ncDHPR mouse model. Out of the three, so far described mutant mammalian DHPR Ca²⁺ channels with ablated Ca²⁺ conducting ability under standard recording conditions, namely R174W (Eltit et al., 2012), E1014K (Lee et al., 2015), and N617D (Dayal et al., 2017), only the voltage-sensor mutant R174W opened partially and produced tail currents under (±)Bay K 8644 administration. This malignant hyperthermia-linked DHPR voltage-sensor mutant R174W also displayed small, but clearly detectable inward Ca²⁺ currents together with enhanced tail currents in response to strong or prolonged depolarizations in the presence of (±)Bay K 8644 (Bannister and Beam, 2013). Integrating previous and present results (Bannister and Beam, 2011), we can conclude that it is impossible to force either of the two DHPR pore mutants, DHPR(N617D) and DHPR(E1014K) into a Ca²⁺ conducting mode by executing the above-described L-type Ca²⁺ current amplifying conditions.

Since both pore mutants, DHPR(N617D) as well as DHPR(E1014K) strictly prevent Ca²⁺ influx even under current enhancing conditions, the striking differences in muscle performance, metabolism, and fiber-type composition between ncDHPR and EK mice (Lee et al., 2015; Georgiou et al., 2015; Dayal et al., 2017) can evidently not be due to DHPR Ca²⁺ conductance. However, the reason for these puzzling phenotypic differences could be attributed to distinct selectivity and permeation properties of physiologically relevant monovalent cations through these mutated DHPRs. Basic biophysical characterization of both the DHPR pore mutants, performed either in the respective mouse model (Lee et al., 2015; Dayal et al., 2017) or in heterologous expression systems (Dirksen and Beam, 1999; Schredelseker et al., 2010; Bannister and Beam, 2011; Beqollari et al., 2018) already pointed out substantial differences in monovalent cation conductance. Specifically, under standard Ca²⁺ current recording conditions with 145 mM Cs⁺ present in the patch pipette to block K⁺ channels (Clay and Shlesinger, 1984), massive outward Cs⁺ currents through DHPR(E1014K) (Bannister and Beam, 2011; Lee et al., 2015; Beqollari et al., 2018) but not through DHPR(N617D) (Schredelseker et al., 2010; Dayal et al., 2017; Beqollari et al., 2018) were observed (see also Figure 1a, top and bottom and Figure 2b, bottom).

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Apparently, the question arose if this Cs⁺ leakiness of DHPR(E1014K) and tightness of DHPR(N617D) is also factual for other monovalent cations like the physiologically relevant Na⁺ ions. To clarify this conundrum, we performed patch-clamp recordings on ncDHPR myotubes under comparable experimental conditions like previously used on DHPR(E1014K) expressed in dysgenic myotubes (Bannister and Beam, 2011).

As demonstrated in Figure 2a and b (top), at near physiological (150 mM) external Na⁺ as the sole monovalent cation and 10 mM external Ca²⁺, no slow inward Na⁺ currents resembling the L-type Ca²⁺ currents were observed in ncDHPR myotubes (n = 8). Even upon reducing external Ca²⁺ from 10 to 1 mM to lower the blocking effect by Ca²⁺, DHPR(N617D) remained fully impermeant to Na⁺ ions (Figure 2a and b, center; n = 9). Both these observations were in stark contrast to the robust slow-activating, non-inactivating inward Na⁺ currents recorded from DHPR(E1014K) under comparable conditions (Bannister and Beam, 2011). The large, rapidly activating and inactivating inward currents observed within the first ~20 ms after the onset of test potentials can be ascribed to endogenous skeletal muscle Na⁺ channel (Na_V) isoforms (Numann et al., 1994) and were to a big extent, present even after administration of 2 µM Na⁺ channel blocker tetrodotoxin (Bannister and Beam, 2011). The outward currents observed in the presence of 150 mM external Na⁺ (Figure 2a and b, top and center) can certainly be ruled out to be Cs⁺ currents through DHPR(N617D) since they are blocked by the K⁺ channel blocker TEA⁺ (Figure 2b, bottom; n = 8) and show kinetics very different from Cs⁺ currents recorded from DHPR(E1014K) (Lee et al., 2015; Bannister and Beam, 2011; Beqollari et al., 2018). Moreover, the current-voltage relationship of these currents which start from approximately −30 mV is linear (10 mM Ca²⁺: R² = 0.99; 1 mM Ca²⁺: R² = 0.97) and this together with TEA⁺ sensitivity strongly points to residual K⁺ currents through the endogenous delayed rectifier K⁺ channel (K_V) (DiFranco and Vergara, 2011). Despite >10 min perfusion with 145 mM Cs⁺ from the patch pipette, these putative K_V currents remained unblocked, probably due to limited diffusion in fairly elongated and narrow myotubes.

To directly test for a putative contribution of DHPR(N617D) in mediating the outward and inward currents described above (Figure 2a and b), we measured whole-cell currents in the presence of the 1,4-DHP Ca²⁺ antagonist nifedipine. As depicted in Figure 2c, patch-clamp recordings performed upon addition of 10 µM nifedipine to the bath solution containing 1 mM Ca²⁺ and 150 mM Na⁺, exhibited nifedipine-insensitive slow outward currents (Figure 2c, left) and rapidly activating and inactivating inward currents (Figure 2c, right). Current-voltage relationship of outward currents (no nifedipine: R² = 0.98; with nifedipine: R² = 0.98) as well as of inward currents (no nifedipine: I_max = −13.22 ± 1.41 pA/pF; n = 6; with nifedipine: I_max = −14.96 ± 1.40 pA/pF; n = 10) were unaffected (p>0.05) by the presence of nifedipine. These results unambiguously confirm that DHPR(N617D) is not accountable for the outward and inward currents observed in the presence of near physiological external Na⁺.

As pointed out above, our results together with previous work (Dirksen and Beam, 1999; Schredelseker et al., 2010; Bannister and Beam, 2011; Lee et al., 2015; Dayal et al., 2017; Beqollari et al., 2018) clearly demonstrate substantial distinct pore properties between DHPR(E1014K) and DHPR(N617D). Although both DHPR pore mutants do not conduct Ca²⁺, DHPR(E1014K) additionally lost its ion-selectivity and robustly conducts monovalent anions like Cs⁺ as well as physiologically relevant Na⁺ and K⁺ even in the presence of physiological concentrations of external Ca²⁺. Although the channel properties of DHPR(E1014K), with its charge conversion of selectivity filter glutamate E₁₀₁₄, are accurately explained by a widely accepted model of cardiac Ca²⁺ channel selectivity and permeation (Yang et al., 1993; Ellinor et al., 1995; Sather and McCleskey, 2003) (see Discussion), the non-conductance mechanism of DHPR(N617D) is still unknown (Schredelseker et al., 2010; Dayal et al., 2017).

Consequently, we wanted to test if the additional negative charge introduced via the N617D substitution, three residues C-terminal to the selectivity filter glutamate in repeat II and positioned towards the pore entrance (Dayal et al., 2017), enhances Ca²⁺ affinity to the pore and resultantly blocks functional Ca²⁺ permeation by hampering the electrostatic repulsion mechanism (Sather and McCleskey, 2003). As a direct index of Ca²⁺ pore-binding affinity assessment, we performed Ca²⁺ block of Li⁺ current experiments on ncDHPR and wt myotubes. In the presence of 100 mM extracellular Li⁺ and without extracellular Ca²⁺ block (free [Ca²⁺]=0), inward Li⁺ currents 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 (Figure 3a). However, increase in extracellular Ca²⁺ concentration to 1 µM showed a highly significant (p<0.001) reduction of Li⁺ currents through ncDHPR (I_max = −0.47 ± 0.10 pA/pF; n = 8) compared to wt (I_max = −1.68 ± 0.25 pA/pF; n = 6) myotubes (Figure 3b). These results indicate a higher efficiency of Ca²⁺ block of Li⁺ currents due to enhanced Ca²⁺ binding affinity to the DHPR(N617D) pore. In particular, at 3 µM external Ca²⁺, no Li⁺ currents could be evoked from ncDHPR myotubes (I_max = −0.06 ± 0.02 pA/pF; n = 7) but small, significant (p<0.001) currents through the wt DHPR (I_max = −0.24 ± 0.04 pA/pF; n = 6) were still existent (Figure 3c). A complete list of peak inward Li⁺ currents at varying free external Ca²⁺ concentrations is presented in Table 1.

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To directly validate if the slow inward Li⁺ currents are conducted by DHPR(N617D), we recorded Li⁺ currents in ncDHPR myotubes in the presence of the 1,4-DHP antagonist nifedipine. As depicted in Figure 4, recordings performed upon addition of 10 µM nifedipine to the bath solution containing 0 Ca²⁺ exhibited a drastic reduction (p<0.001) of slow inward Li⁺ currents (no nifedipine: I_max = −2.41 ± 0.27 pA/pF; n = 11; with nifedipine: I_max = −0.35 ± 0.13 pA/pF; n = 16). These results confirm that the slow inward Li⁺ currents observed in the absence of external Ca²⁺ are mediated by DHPR(N617D).

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Large, rapidly activating and inactivating inward currents detected in both wt and ncDHPR myotubes within the first ~20 ms of the onset of test potentials are Li⁺ currents through endogenous skeletal muscle Na⁺ channels, Na_V (Numann et al., 1994; DiFranco and Vergara, 2011). Interestingly, their amplitudes appear to correlate negatively to the slow Li⁺ current amplitudes through wt or mutant N617D DHPRs at different external Ca²⁺ concentrations (Figure 3a-c) and were similarly amplified upon nifedipine block of DHPR(N617D) channels at external free [Ca²⁺]=0 (Figure 4b). A possible competition between Ca_V and Na_V channels for Li⁺ ions, with Ca_V taking the priority was not investigated further in the present study.

Eventually, to quantify the impact of the DHPR pore mutation N617D on Ca²⁺ pore binding-affinity in comparison to wt DHPR, Ca²⁺ concentration-response curves displaying inhibition of peak inward Li⁺ currents by Ca²⁺ were analyzed by a nonlinear fit with variable slope (four parameter). As demonstrated in Figure 3d, Ca²⁺ block of inward Li⁺ currents through the skeletal muscle wt DHPR displays an IC₅₀ of 1.57 µM (95% CI: 1.36–1.80 µM), which is highly comparable to published values of cardiac DHPR Ca²⁺ pore binding affinity (Yang et al., 1993; Ellinor et al., 1995; Cibulsky and Sather, 2000; Sather and McCleskey, 2003). Interestingly, pore mutant DHPR(N617D) exhibited an IC₅₀ of 372.8 nM (95% CI: 334.4–415.8 nM) which is indeed 4.2-fold shifted to lower Ca²⁺ concentrations. Thus, in the mutant DHPR(N617D), introduction of the negatively charged residue D₆₁₇ into the DHPR pore in close vicinity of the selectivity filter EEEE results in a significant (p<0.01) decrease in IC₅₀ from µM to nM concentrations. Notably, also the Hill coefficient (n_H) was significantly (p<0.01) different between wt DHPR (−3.32; 95% CI: −4.68 – −2.61) and DHPR(N617D) (−1.39; 95% CI: −1.59 – −1.23) (see Discussion). Since atypical high-affinity binding of Ca²⁺ to the mutant pore is apparently incompatible with Ca²⁺ conductance, this supports the idea of a mechanism by which the mutant DHPR(N617D) pore is occluded.

In an attempt to answer the above questions, we intend to expand a widely accepted molecular model of Ca²⁺ channel selectivity and permeation based on two elegant studies from the Tsien lab (Yang et al., 1993; Ellinor et al., 1995), and comprehensively discoursed in the review of Sather and McCleskey, 2003. According to this model, one Ca²⁺ ion binds to a single high-affinity site formed by all four glutamates (EEEE locus) of the DHPR selectivity filter. This tight embracement of Ca²⁺ in the DHPR pore is a prerequisite for the high selectivity for Ca²⁺ over Na⁺, K⁺, or other monovalent cations. However, to enable rapid passage of Ca²⁺ through the pore, a two-site mechanism that overcomes this tight Ca²⁺ binding is essential. Accordingly, the EEEE locus has been suggested to be physically flexible. Hence, irrespective that all four selectivity filter glutamates are needed to hold a single Ca²⁺ ion with high affinity (K_D ~1 µM), their conformation can rapidly rearrange to accommodate a pair of Ca²⁺ ions within the pore, but then both bound with much lower affinity (apparent K_D ~14 mM). This intermediate short-lived low-affinity state, together with a Ca²⁺- Ca²⁺ repulsion mechanism occurring in this doubly occupied pore, whereby one of the occupying Ca²⁺ ions is pushed out to the cytosolic side, is the basis for fast Ca²⁺ ion passage through the pore.

Although the Ca²⁺ selectivity filter in form of the conserved EEEE locus within the pore of high threshold voltage-gated Ca²⁺ channels (HVA VGCC) satisfactorily explains divalent/monovalent ion selection, it neither explains the differences in the selectivity for Ca²⁺ among other divalent ions nor the observed distinct conductances through the different HVA VGCC isoforms (Cens et al., 2007). In their interesting study, Cens et al., 2007 via point mutational analyses and molecular modeling identified a ring of non-conserved negatively charged residues located at homologous positions in each of the four repeats of the DHPR pore, which were responsible for the distinct channel profiles. This ring coined as ‘divalent cation selectivity’ (DCS) locus, is present in different constellations in every VGCC and is located towards the outer channel pore region in close vicinity of the selectivity filter EEEE locus. The DCS locus might constitute an additional, low-affinity Ca²⁺-binding site which, together with distinct negative charges closely adjacent to the EEEE locus (Williamson and Sather, 1999), plays a crucial role in defining and directly participating in the generation of different Ca²⁺ conductances in different HVA Ca²⁺ channels (Cens et al., 2007).

As discussed above, proper Ca²⁺ channel permeation and high selectivity are essentially dependent on a single high-affinity Ca²⁺-binding site formed by all four glutamates of the DHPR selectivity filter to assure tight embracement of Ca²⁺. Any substitution in the EEEE locus abolishes/decreases this high (µM) Ca²⁺ pore binding affinity as demonstrated by Ca²⁺ block of Li⁺ current experiments (Yang et al., 1993; Ellinor et al., 1995; Sather and McCleskey, 2003). Specifically, the strongest impact on the binding affinity was produced by exchange of E in repeat III. The EIIIK mutation drastically reduced the pore’s affinity for Ca²⁺ to 1000-fold, as is depicted by an increase in IC₅₀ from ~1 µM to ~1 mM for Ca²⁺ block of I_Li+ (Yang et al., 1993). Although these classical affinity experiments where performed in the cardiac DHPR, the comprehended selectivity/conductance model appears to be congruent with the skeletal muscle DHPR. Accordingly, the large outward Cs⁺ current found in the skeletal muscle EIIIK mutant DHPR(E1014K) (Bannister and Beam, 2011; Lee et al., 2015; Beqollari et al., 2018), which was not blocked even in the presence of 10 mM external Ca²⁺, was consequently interpreted as an indication of very little residual Ca²⁺ binding within the DHPR(E1014K) pore (Dirksen and Beam, 1999; Beqollari et al., 2018). Similarly, a considerable inward Na⁺ current through EK myotubes despite external Ca²⁺ concentration as high as 10 mM (Bannister and Beam, 2011) again indicates a very marginal, low-affinity binding of Ca²⁺ within the DHPR(E1014K) pore. Consequently, low-affinity pore-bound Ca²⁺ is unable to block the flux of any cation in both directions and hence Ca²⁺ selectivity is abolished in the mutant DHPR(E1014K). In addition, since the EEEE locus is mutated to EEKE, attraction of a second Ca²⁺ and subsequent competition for binding valences with the Ca²⁺ ion that is already bound with low affinity to this EEKE locus is impossible. Absence of this intermediate doubly occupied pore and thus, of the Ca²⁺- Ca²⁺ repulsion mechanism as the basis for fast, unidirectional Ca²⁺ ion passage through the pore is sufficient to explain the lack of Ca²⁺ conductance through the mutant DHPR(E1014K).

Now the question arose, how to understand the pore blocking mechanism observed in DHPR(N617D) by coalescing the models discussed above? Figure 5a depicts the putative mechanism of Ca²⁺ conductance through wt DHPR. The carboxyl oxygens of the DCS locus point toward the pore lumen, allowing coordination of incoming divalent cations with a preference for Ca²⁺ (Cens et al., 2007). According to our postulated pore model (Figure 5a), Ca²⁺ ions from the t-tubular (extracellular) side are attracted to the negative charges of the DCS locus, which in mouse DHPRα_1S is formed by D₂₉₆ of repeat I, E₁₃₂₇ of repeat IV, and supported by D₆₁₅ of repeat II. This loosely bound Ca²⁺ ion is easily mobilized (probably by charge repulsion from excess Ca²⁺ ions in the t-tubule) and migrates deeper into the pore to compete with the tightly bound Ca²⁺ ion for binding valences of the EEEE locus (in mouse skeletal-muscle DHPR: E₂₉₂, E₆₁₄, E₁₀₁₄, E₁₃₂₃). Henceforth, due to the reduced binding (µM to mM affinity), Ca²⁺- Ca²⁺ repulsion (Sather and McCleskey, 2003) takes place, eventually pushing the loosely bound Ca²⁺ into the cytosol. This conceptual model is supported by simulation experiments as depicted in Figure 6. Molecular dynamics simulations show that the EEEE locus attracts and stabilizes a single Ca²⁺ ion (Figure 6b–c). However, in the wt DHPR we also observe conformational changes in the EEEE locus that allow binding of a second Ca²⁺ ion. This additional Ca²⁺ ion results in a weaker binding of the glutamate residues to both Ca²⁺ ions, thereby causing a repulsion between the two ions, which is reflected in their decreasing distance to as low as 6 Å (Figure 6c, left). Furthermore, metadynamics simulations show that as a consequence of this Ca²⁺ - Ca²⁺ repulsion occurring in the doubly occupied EEEE locus, one of the two Ca²⁺ ions moves toward the cytosolic side (Figure 6c, left; Figure 6—video 1). The weaker binding of the Ca²⁺ ions to the EEEE locus of the wt DHPR compared to the mutant DHPR(N617D), is reflected in the significantly (p<0.001) lower free energy barrier (Figure 6d).

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Contrary to this smooth Ca²⁺- conducting mechanism of wt DHPR, the additional negative charge D₆₁₇ in mutant DHPR(N617D), introduced in the close vicinity to the residue D₆₁₅ in repeat II (Figure 5b), creates an additional binding valence and as a result induces an aberrant high Ca²⁺ binding-affinity to the DCS locus. According to our model, this considerably tighter bound Ca²⁺ is consequently not sufficiently mobile anymore to travel deeper into the pore to compete for the binding valences of the selectivity-filter EEEE locus with the already strongly bound Ca²⁺ ion. Overall, lack of formation of the intermediate short-lived lower-affinity Ca²⁺ binding state, together with the consequential lack of Ca²⁺- Ca²⁺ repulsion at the EEEE locus explicitly explains the absence of Ca²⁺ influx through the DHPR(N617D) pore. Congruently, molecular dynamics simulations show that immediately after the equilibration step, one Ca²⁺ ion is stabilized at the EEEE locus while the other Ca²⁺ is bound to the DCS locus (Figure 6c, right; Figure 6—video 2). This translocation of the Ca²⁺ ions to the DCS and EEEE locus occurs already within 1 ns of simulation time succeeding the last step of the equilibration protocol. Here, the distance between the two Ca²⁺ ions is ~9 Å. The strong binding of the two Ca²⁺ ions to the EEEE and DCS locus makes it impossible for any other ion, like Li⁺, to pass through the DHPR(N617D) pore. Thus, simulations of pulling of Ca²⁺ ions through the selectivity filter of mutant DHPR(N617D) result in a significantly (p<0.001), ~8 times higher energy barrier compared to wt DHPR (Figure 6d), which is in accordance with the experimentally observed complete occlusion of the DHPR(N617D) pore in the presence of physiological concentrations of extracellular Ca²⁺ ions (Figure 3). This rather static condition in the DHPR(N617D) pore is well expressed in its lower Hill slope compared to wt DHPR (see Figure 3d). The Hill slope/Hill coefficient (n_H) derived from four parameter logistic fit of dose-response curve is best portrayed as an ‘interaction’ coefficient, reflecting the extent of cooperativity among multiple binding sites (Prinz, 2010). The considerably more dynamic Ca²⁺ interactions in the wt DHPR pore with its successive short-lived intermediate high and low binding affinities and repulsion mechanisms are consequently apparent in the higher n_H compared to DHPR(N617D).

Point mutation N617D implemented for the creation of mouse model ncDHPR (Dayal et al., 2017) was originally identified to be responsible for DHPR Ca²⁺ non-conductivity in zebrafish fast (glycolytic/white) skeletal muscle (Schredelseker et al., 2010). Additionally, with studies on the low-Ca²⁺ conducting DHPR of sterlet (Acipenser ruthenus), which is phylogenetically somewhere in between mouse and zebrafish, we showed (Schrötter et al., 2017) that during vertebrate evolution (i.e. from the mammalian species, e.g. mouse, to the teleost fishes, e.g. zebrafish) a steady loss of DHPR Ca²⁺ conductance occurred. Subsuming results of several studies, we proposed the hypothesis that during evolution from mammals to teleost fishes an accumulation of DHPR amino acid exchanges occurred that contributed to the reduction of Ca²⁺ conductance (Schredelseker et al., 2010; Dayal et al., 2017; Schrötter et al., 2017). Mutation N→D (N617D; mouse numbering) that finally ‘turned off’ the already reduced Ca²⁺ conductance evolved only in quite a late phylogenetic stage (Dayal et al., 2017; Schrötter et al., 2017), following the teleost-specific third round (Ts3R) of gene duplication (Meyer and Van de Peer, 2005; Glasauer and Neuhauss, 2014). Beside DHPR non-conductivity, the evolutionary pressure that caused additional substantial modifications in skeletal muscle organization and physiology in teleost fishes (Schredelseker et al., 2010; Dayal et al., 2017; Schrötter et al., 2017) arose from the critical demand for tighter controlled, faster and stronger muscle contractions, crucial for high-speed movements in the aquatic prey-predator environment (Dayal et al., 2019).

Interestingly, Ts3R headed into the evolution of a second DHPR isoform in zebrafish slow (oxidative/red) skeletal muscle that is likewise Ca²⁺ non-conducting (Schredelseker et al., 2010). This slow muscle DHPR is so far the only described innate DHPR with a distorted EEEE locus, where glutamate of repeat I is substituted by glutamine. Exchange of this selectivity filter E₂₉₂ with Q in a GFP-tagged rabbit DHPRα_1S clone (Grabner et al., 1998) yielded mutant DHPR(E292Q), which upon heterologous expression in dysgenic myotubes confirmed the abolishment of inward Ca²⁺ currents (Schredelseker et al., 2010) with a slight outward Cs⁺ current, typically starting at +20 to+30 mV (Bannister and Beam, 2011). As described earlier (Yang et al., 1993), the EQ pore mutation in repeat I of the cardiac DHPR exerted a minor effect, as the increase in IC₅₀ was only twofold compared to the wt. If we assume that a similar right-shift of affinity also holds true for the skeletal muscle DHPR(E292Q), then appropriate Ca²⁺ pore-affinity essential for proper Ca²⁺ selectivity and Ca²⁺ conductance must exist in a surprisingly small range. Incorporation of our present and previously published data (Yang et al., 1993; Schredelseker et al., 2010) indicates that this small range might be within approximately one order of magnitude, somewhere between 0.37 (IC₅₀ for N617D) and 3.2 µM (2-fold IC₅₀ for wt). The hampered Ca²⁺ selectivity and conductance mechanism of mutant DHPR(E292Q) (Figure 5—figure supplement 1a) is expected to be essentially the same as discussed above for DHPR(E1014K). In brief, low-affinity Ca²⁺ binding to the QEEE locus (Figure 5—figure supplement 1a) cannot support the crucial Ca²⁺ - Ca²⁺ repulsion mechanism and thus, Ca²⁺ conductance through mutant DHPR(E292Q) is blocked. Likewise, Ca²⁺ block of the bidirectional flux of monovalent cations, and hence Ca²⁺ selectivity is abolished.

Lastly, a third evolutionary concept also yielding a Ca²⁺ non-conducting DHPR was identified in the fast skeletal muscle of teleost fishes (Schredelseker et al., 2010). Although in the early phylogenetic teleost species (including zebrafish from the order cypriniformes) mutation N→D (N617D, mouse numbering) is the archetypical mutation to block DHPR Ca²⁺ influx, in phylogenetically higher developed teleost species starting with the order lophiiformes (anglerfishes), this negatively charged D was lost by mutating to a neutral T (Schredelseker et al., 2010). Concurrent to this D→T mutation, DHPR Ca²⁺ non-conductivity was re-installed by mutation of another D, which is one of the negative charges in the DCS locus (located in pore repeat I) and highly homologous in all mammalian L-type Ca²⁺ channels, to positively charged K (D→K). As demonstrated previously (Schredelseker et al., 2010), exchange of this DCS locus D₂₉₆ with K in a GFP-tagged rabbit DHPRα_1S clone yielded mutant DHPR(D296K). Upon heterologous expression in dysgenic myotubes, this single charge conversion was sufficient to abolish inward Ca²⁺ currents. According to our combined model of Ca²⁺ selectivity and conductance and illustrated in Figure 5—figure supplement 1b, K₂₉₆ does not permit formation of an active DCS locus, and thus Ca²⁺ from the t-tubular (extracellular) space is no more attracted to the DCS locus. Resultantly, there is lack of easy to mobilize low-affine DCS-bound Ca²⁺ that would compete with the tightly EEEE-bound Ca²⁺ for the binding valences of the EEEE locus (Figure 5—figure supplement 1b). Thus, the Ca²⁺- Ca²⁺ repulsion mechanism (Sather and McCleskey, 2003) and pushing out of the Ca²⁺ bound to the selectivity filter into the cytosol cannot take place. The surprising implication of charge conversion D296K in blocking of inward DHPR Ca²⁺ flux proves the importance of the DCS locus for proper inward DHPR Ca²⁺ currents in skeletal muscle and consequently, fundamentally supports our model of DHPR Ca²⁺ selectivity and Ca²⁺ conductivity.

Generation of the Ca²⁺ non-conducting (nc)DHPR knock-in mouse strain, carrying a point mutation in the Cacna1s gene coding for N617D in pore loop II was described previously (Dayal et al., 2017). Animal breeding, care and maintenance was conducted in compliance with the guidelines of the EU Directive 2010/63/EU and approved by the Austrian Ministry of Science (BMWF-5.031/0001-II/3b/2012). Mice were housed in a controlled environment with a 12/12 hr light/dark cycle and had access to food and water ad libitum.

Primary myoblasts from new born up to 4-day-old pups homozygous for the non-conducting L-type Ca²⁺ channel mutant DHPR(N617D) or wild-type channel were enzymatically isolated and cultured in a humidified 37°C incubator with 5% CO₂ as described previously (Dayal et al., 2017). Myotubes were maintained in growth medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 10% horse serum, 25 mM HEPES, 4 mM L-glutamine, and 1x penicillin/streptomycin and later replaced with differentiation medium (no fetal calf serum and only 2% horse serum).

Ionic currents were evoked by a standard 200 ms voltage-step protocol from −50 to +80 mV in 10 mV increments from a holding potential of −80 mV (Dayal et al., 2017), unless otherwise stated. To reduce inward currents via endogenous Na_V and T-type Ca²⁺ channels, every test pulse was preceded by a 1 s prepulse to −30 mV followed by a 50 ms repolarization to −50 mV (Adams et al., 1990). Borosilicate glass patch pipettes had resistance of 2–3 MΩ when filled with (in mM) 145 Cs-aspartate, 2 MgCl₂, 10 HEPES, 0.1 Cs₂-EGTA, and 2 Mg-ATP (pH 7.4 with CsOH). The standard bath solution for recording Ca²⁺ currents contained (in mM): 10 CaCl₂, 145 TEA-Cl and 10 HEPES (pH 7.4 with TEA-OH). Myosin-II blocker BTS (100 µM, Sigma) was constantly present in the bath solution.

To test if depolarization-induced potentiation protocols known to promote mode two gating in L-type Ca²⁺ channels could evoke currents through DHPR(N617D), strong or long depolarizations in the presence of racemic 1,4-dihydropyridine (DHP) agonist (±)Bay K 8644 (10 µM) were performed (Bannister and Beam, 2011; Bannister and Beam, 2013). Pulse protocol for strong depolarization is depicted in Figure 1b. Briefly, 200 ms depolarization to either +90 mV or +60 mV is followed by a +60 mV pulse for 100 ms and finally by a repolarization to −20 mV for 70 ms. For long depolarization (Figure 1c), prolonged 2 s pulses from +10 - +80 mV in 10 mV increments were applied starting from a holding potential of −80 mV with an intermediate repolarizing step to −50 mV.

To investigate if DHPR(N617D) conducts slow-activating, non-inactivation inward Na⁺ currents, 145 mM TEA-Cl in standard bath solution was replaced by 145 mM NaCl (pH 7.4 with NaOH) to achieve near physiological Na⁺ concentration (150 mM). Furthermore, to test if these Na⁺ currents were also subject to block by Ca²⁺, 10 mM Ca²⁺ was reduced to near physiological 1 mM Ca²⁺.

To assess Ca²⁺ pore-binding affinity, dose-inhibition experiments for Ca²⁺ block of inward Li⁺ currents were performed. The bath solution for recording Li⁺ currents contained (in mM): 100 LiCl, 10 HEPES, 10 EGTA, and 25 for CaCl₂ plus TEA-Cl (pH 7.4 with TEA-OH). Desired free Ca²⁺ concentrations (0 to 30 µM) were obtained by calibrating CaCl₂ and TEA-Cl concentrations calculated using the MaxChelator simulation program (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/) (Supplementary File Table 1).

To test if the inward Li⁺ currents under external free [Ca²⁺]=0 as well as the slow outward and fast inward currents recorded under external 150 mM Na⁺ and 1 mM Ca²⁺ are mediated by DHPR(N617D) in ncDHPR myotubes, 10 µM of the 1,4-DHP antagonist nifedipine was added to the respective bath solutions.

All recordings were performed at room temperature using the Axopatch 200B amplifier (Axon Instruments Inc, CA), filtered at 1 kHz and sampled at 5 kHz.

Data were analyzed and plotted using ClampFit (v10.7; Axon Instruments), SigmaPlot (v11.0; Systat Software, Inc) and Prism 8 (GraphPad Software, LLC). Data are represented as mean ± SEM and n = number of myotubes. Statistical significance was calculated using unpaired Student’s t-test, unless otherwise stated and was set as follows: *p<0.05, **p<0.01, and ***p<0.001.

Atomic models were based on the cryo-EM structure of the rabbit DHPRα_1S - verapamil complex with a dilated intracellular gate associated to the binding of the phenylalkylamine Ca²⁺ antagonist drug verapamil (PDB accession number 6JPA) (Zhao et al., 2019). The structure of mutant DHPR(N617D) was derived from wt DHPR structure by replacing N₆₁₇ with the negatively charged residue D₆₁₇ and carrying out a local energy minimization using MOE (Molecular Operating Environment, Chemical Computing Group, version 2020.01). For simulations, we removed the voltage-sensing domains and truncated the S5 and S6 helices of each repeat, keeping the last nine residues of the S5 and S6 helices. The C- and N-termini of each repeat were capped with acetylamide (ACE) and N-methylamide to avoid perturbations by free charged functional groups. The starting structures for simulations were prepared in MOE using the Protonate3D tool (Labute, 2009). To neutralize the charges, we used the uniform background charge (Case et al., 2020; Hub et al., 2014). Using the tleap tool of the AmberTools20 package (Case et al., 2020; Roe and Cheatham, 2013), crystal structures were soaked in cubic water boxes of TIP3P water molecules with a minimum wall distance of 10 Å to the protein (Jorgensen et al., 1983; El Hage et al., 2018; Gapsys and de Groot, 2019). We added a total of 10 Ca²⁺ ions, corresponding to a concentration of approximately 10 nM. For all simulations, parameters of the AMBER force field 14 SB were used (Maier et al., 2015). The structures were carefully equilibrated using a multistep equilibration protocol (Wallnoefer et al., 2011).

For both wt DHPR and mutant DHPR(N617D), 10 ns of molecular dynamics (MD) simulations were performed in an isothermal - isobaric (NpT) ensemble using the GPU MD simulation engine pmemd.cuda (Salomon-Ferrer et al., 2013) to further equilibrate the structures in the presence of the Ca²⁺ ions. Bonds involving hydrogen atoms were restrained by applying the SHAKE algorithm (Miyamoto and Kollman, 1992), allowing a time step of 2 fs. Atmospheric pressure of the system was preserved by weak coupling to an external bath using the Berendsen algorithm (Berendsen et al., 1984). The Langevin thermostat (Doll et al., 1975; Adelman, 1976) was used to maintain the temperature at 300 K during simulations.

Metadynamics is a powerful method to explore the properties of multidimensional free energy landscapes and to enhance the sampling of configurational space in reasonable computing time (Barducci et al., 2011). Metadynamics reconstructs the free energy surface as a function of few selected degrees of freedom, referred to as collective variables (CV), which accelerate rare events in the systems. The CVs should be able to characterize the key features of physical behavior of interest, distinguish between all different metastable states, and include the slow degrees of freedom. In metadynamics, an external history-dependent repulsive bias potential function constructed as a sum of Gaussians is deposited along the trajectory in the CV space and thereby, discourages revisiting and oversampling of same configurations. For metadynamics simulations, we used the GROMACS version 2019.2. The aim of the metadynamics simulation was to capture the movement of Ca²⁺ ions along the selectivity-filter conducting pathway and their passing through the EEEE motif. As CV, we chose the distance between the center of masses (COM) of the EEEE motif residues and the upper Ca²⁺ ion.

Simulations were performed at 300 K in an NpT ensemble. We used a Gaussian height of 1.5 kJ/mol and width of 0.1 nm. For both wt DHPR and mutant DHPR(N617D), five repetitions of metadynamics runs, each 10 ns, were performed. Pymol Molecular Graphics System was used to visualize the key interactions and differences between wt DHPR and mutant DHPR(N617D) pore conductances.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This study was supported by the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung, FWF) Research grants P23229-B09 (to M.G.), P27392-B21 (to MG and AD).

1. Received: September 24, 2020
2. Accepted: May 28, 2021
3. Accepted Manuscript published: June 1, 2021 (version 1)
4. Version of Record published: June 7, 2021 (version 2)
5. Version of Record updated: June 11, 2021 (version 3)

© 2021, Dayal et al.

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