Non-ionotropic voltage-gated calcium channel signaling

Figures & data

Figure 1. Excitation transcription coupling is critically dependent on Ca²⁺ binding to the selectivity filter. (a) A proposed structural model of the EEEE motif, four glutamate comprising high-affinity Ca²⁺ binding site of the channel ion pore, according to Lipkind and Fozzard [62] (upper, left). A single point selectivity filter mutant of the Ca²⁺-impermeable channel;, α₁1.2^L745P/E363A (upper, middle), and a double point selectivity filter mutant of the Ca²⁺-impermeable channel, α₁1.2^{L745P/E363A/E1115} mutant (upper, right) (b) HEK293 cells expressing the wt, impermeable mutant or the pore mutants stimulated by 70 mM KCl for 3 min. Phosphorylation of ERK1/2, RSK, and CREB monitored after stimulation and c-Fos expression, 60 min later, monitored by western blot analysis. Quantification plotted as averages (±SEM) of 3 independent experiments normalized to the corresponding non-phosphorylated proteins using the corresponding antibodies. All experiments were done in triplicate transfections and performed 3 times using different cell batches. One-way ANOVA was performed to determine statistically significant differences for K70-stimulated cells. *p < 0.05, **p < 0.01, ***p < 0.001. Adapted from [23].

Excitation transcription (ET) coupling in HEK293 cells expressing Cav1.2 is triggered via the Ras/ERK/CREB pathway. ET coupling is triggered also by a single-point calcium impermeable channel mutant, indicating noninotropic mediated transcriptional activity. Inhibition of ET coupling by mutations in the selectivity filter of this Ca2+ impermeable channel underscores the critical role of ion pore occupancy in ET coupling.

Figure 2. The IQ motif of Cav1.2, Ca²⁺/CaM, or calcineurin, are not involved in ET coupling. (a) Schematic view of the IQ calmodulin (CaM)-binding motif location at the C-tail of the α₁1.2 subunit. (b) The contribution of IQ motif to ET coupling HEK293 cells transfected with wt Cav1.2 (α₁1.2/β2b/α2δ) or the IQ Cav1.2 mutant (α₁1.2^I1624A/β2b/α2δ) treated 72 hr later with non-depolarizing (2.5 mM KCl; basal) or depolarizing (70 mM KCl; dep) solutions for 3 min. Phosphorylation of ERK, RSK, and CREB was detected using the corresponding anti-phospho-protein antibodies (upper) and quantified normalizing with antibodies of the corresponding non-phosphorylated proteins and anti α2δ subunit antibodies (lower). (c) The effect of cyclosporine a (CsA) the selective CaN inhibitor, and trifluoperazine (TFP), a Ca²⁺/CaM inhibitor, on ET coupling HEK293 cells transfected with wt Cav1.2 (α₁1.2/β2b/α2δ) or the IQ mutant (α₁1.2^I1624A/β2b/α2δ). Seventy-two hr after transfection the cells were treated with 100 nM CsA or 10 μM TFP for 2 hr, the cells were pulsed with 2.5 mM KCl; basal, or 70 mM KCl; dep, solutions for 3 min. One-way analysis of variance (ANOVA) was used to determine statistically significant differences. The plotted values of net phosphorylation are averages (±SEM) of three independent experiments normalized to the corresponding non-phosphorylated proteins. Adapted from [23].

Excitation transcription (ET) coupling in HEK293 cells expressing Cav1.2 is triggered via the Ras/ERK/CREB pathway and is not affected by Ca2+ dependent reactions. ET coupling is not affected by mutating the calmodulin (CaM) binding site (IQ motif), inhibition of Ca2+/CaM or calcineurin (CaN). These data indicates no contribution of Ca2+ entry and involvement of CaM activity in ET coupling via this pathway.

Figure 3. The Non-ionotropic activity of Cav1.2 mediates excitation transcription (ET) coupling. A schematic illustration of VGCC in the closed state, occupied with a single Ca²⁺ ion tightly bound (<1 µM) to the EEEE motif (left). Upon arrival of an action potential (XXXv) the open channel now occupied with an additional Ca²⁺ ion triggers transcription of immediate early genes (IEGs), also known as Primary-Response-Genes (rPrgs), prior to and independent of ion flow (non-ionotropic activity) (middle). Subsequent Ca²⁺ influx (ionotropic activity) and elevation of [Ca²⁺]_i might suggest transcription of delayed Primary-Response-Genes (dPrgs), either via the CaM-Ca²⁺ dependent pathway and/or other intracellular Ca²⁺ dependent processes (right).

A schematic illustration of VGCC mediating ET coupling in two distinct steps. In the inactive closed state, the selectivity filter of the channel is occupied with a single calcium ion. Upon the arrival of an action potential, conformational changes occur simultaneously with channel opening, facilitating the binding of additional calcium ions to the selectivity filter. These conformational changes preceding the influx of Ca2+, involve transcription of immediate early genes (IEGs), also known as Primary-Response-Genes (rPRGs) in a non-ionotropic manner. The subsequent ionotropic step involves Ca2+ entry, mediating transcription of delayed Primary-Response-Genes (dPRGs), either via the CaM-Ca2+ dependent pathway and/or other intracellular Ca2+ dependent processes.

Figure 4. Intra-membrane signaling between the voltage-gated Ca²⁺ channel and syntaxin 1A coordinates synchronous release. (a) Botulinum C1 (BotNT/C1) cancels the effect of syntaxin 1A (Sx1A) on Cav1.2 current amplitude. Expression of Sx1A with Cav1.2 with or without BotNT/C light chain in Xenopus oocytes injected with cRNA of α₁1.2/β2A/α2δ subunits. At day 2 after injection with the channel subunits, oocytes were injected with cRNA Sx1A, and at day 4 with cRNA BotNT/C light-chain. Protein expression at day 6 was monitored by western blot with anti Sx1A antibodies(left). Ca²⁺ currents were evoked from a holding potential of −80 mV by a single voltage step of 140 ms duration to a step potential of +20 mV. Depolarization triggered leak-subtracted peak current-voltage relationship (see collected data from oocytes expressing α₁1.2/β2a/α2δ subunits alone (open circle), with Sx1A (closed circle), with Sx1A and BotNT/C (square) (middle); (Student’s t-test, p < 0.01 (n = 6–9). Adapted from ref [83]. Schematics of Cav1.2 in a complex with BotNT/C cleaved Sx1A (right); plotted to concur with literature reports, for example, refs [83,88]. (b) Mutating Cys-271 and Cys-272 of the Sx1A TMD disrupts Sx1A interaction with Cav1.2. Oocytes were injected with α₁1.2/α₂δ/β2a and at day 2 with Sx1A or Sx1A double mutant Sx1A^CC/VV. at day 6 after injection, Ca²⁺ currents were evoked from a holding potential of −80 mV by a single voltage step of 140 ms duration to a step potential of +20 mV in oocytes expressing the three channel subunit, with and without Sx1A or Sx1A^CC/VV (left). Leak-subtracted peak current-voltage relationship (see inset): collected data from oocytes expressing the three channel subunits (open circle) together with Sx1A (closed circle) or Sx1A^CC/VV (open rectangle) (middle).The data points correspond to the mean ± S.E. of current amplitude (n = 8). Adapted from [89]. Schematic presentation of Cav1.2 in a complex with the Sx1A TMD mutant (Sx1A^CC/VV), (right); plotted to concur with literature reports [90]. (c) Dominant inhibitory effect of Sx1A^CC/VV transmembrane mutant on depolarization-evoked catecholamine release in bovine chromaffin cells Amperometry traces for control, GFP-infected, RFP-wt Sx1A infected, and RFP-Sx1A^CC/VV infected cells elicited by a pulse of 60 mM KCl (K60), indicated by the arrow. (d) Cumulative spike counts plotted versus time illustrate the time course of catecholamine secretion triggered by membrane depolarization (starting at t = 10; Left). Expanded view of the initial cumulative spike counts shown in (c) (right). (e) Average number of spikes elicited per cell (Left). The total mean charge represents total CA secretion, which is the average area underneath the spikes and is presented as the percentage of average secretion per cell (Middle), the mean frequency of the initial rate was calculated as the maximum slope in plot (b) (right) during the first 30 sec of recording **p < 0.005 Adapted from ref [79].

Modification at the channel/Sx1A interface resulting from mutations of two highly conserved Cys residues within the transmembrane domain (TMD) of Sx1A, or through cleavage of Sx1A by BotNT/C, demonstrate a distinct correlation with suppression of evoked release. This direct transmembrane signaling mechanism between the channel and exocytotic machinery signifies a functional protein-protein-interaction-based mechanism, which potentially support conformation-triggered exocytosis.

Figure 5. Oxidation of Sx1A disrupts the α₁1.2/Sx1A intracellular signaling and blocks evoked-transmitter release. (a) Representative superimposed α₁1.2/β2/α2δ current traces (Cav1.2) in the absence and in the presence of AuF (left); Sx1A/Sx1A + AuF as indicated (middle) or Sx1A+AuF+AD4 (right). (b) Leak subtracted current-voltage relationships of α₁1.2/β2/α2δ expressed either alone (open circles) or with AuF (open rectangle left); Sx1A/Sx1A + AuF (●/,^▴ middle) or Sx1A+AuF+AD4 (^◂, right). Inward Ca²⁺-currents evoked from a holding potential of −80 mV to various test potentials in response to 200 ms test pulse at 5 mV increments. (c) G/Gmax values as indicated in (b) The data points correspond to the mean ± SEM of currents (n = 8–15). (d) Representative amperometric current traces elicited by a puff of K60 in control cells, AuF-treated cells (5 μM; 30 min), and cells treated with AuF followed by CB3 for 30 min at the indicated concentrations (e) Cumulative events per cell plotted versus time; an expanded view of the initial cumulative spike counts (right) and the mean frequency of the initial rate (left) (f) Reversal of AuF-induced inhibition of CA release by TXM-CB3. CA secretion elicited by K60 in control cells, AuF-treated cells (5 μM; 30 min), or cells pretreated with AuF followed by TXM-CB3 for 30 min, at the indicated concentrations. The initial rates of CA secretion during the first 20s of recording (left), total CA secretion (middle), and spikes per cell (right) (g) CA secretion elicited by K60 in control cells, AuF-treated cells (5 μM; 30 min), or cells pretreated with AuF followed by NAC-amide (AD4) for 30 min, at the indicated concentrations. The initial rates of CA secretion during the first 20s of recording (left), total CA secretion (middle), and spikes per cell (right). Total secretion calculated as the average picocoulombs at each spike and sustained cumulative spike count. The mean frequency of the initial rate was calculated as the maximum slope during the first 20s of recording. Means were calculated for individual cells as an average of more than 500 spike events adapted from [79,94].

The oxidation effect of a thiol-oxidizing reagent on Cav1.2/Sx1A currents and on evoked-release in bovine chromaffin cells is fully reversed by thiol reducing reagents such as thioredoxin mimetic (TXM) peptides CB3 or NAC-amide (AD4/NACA). These results also correlate with voltage-clamp Xenopus oocyte studies, in which phenylarsine oxide (PAO), a selective Cys vicinal oxidizing thiol reagent, disrupts Sx1A interplay with the channel, and is fully reversed by a reducing reagent. These findings suggest that the reversible redox sensitivity of the exocytotic event correlates with the interaction between Sx1A and VGCC, indicating a direct modulation of channel activity by the redox state of Sx1A.

Figure 6. Impermeable cation or calcium-impermeable channel support depolarization evoked secretion. (a) Catecholamine release monitored by amperometry using carbon fiber electrode brought in close proximity of a single bovine chromaffin cell (b) Traces of amperometric currents elicited in bovine chromaffin cells by a 10-s pulse of 60 mM KCl (K60) in the absence and presence of 5 μM nifedipine (Nif) using 2 mM Ca²⁺ as a charge carrier, or in Ca²⁺-free medium (upper). Cumulative events per cell plotted versus time (lower, left); number of spikes per cell following stimulation shown as mean ± SE; in the presence (n = 27) or in the absence (n = 36) of 5 μM Nif (lower right). (c) Traces of amperometric currents triggered by K60 in the presence or in the absence of 5 μM Nif, using 0.2 mM La³⁺ in nominally Ca²⁺ free solution (left). Cumulative events plotted versus time (middle) and the number of spikes per cell as mean ± SE in the presence (n = 16) or in the absence (n = 31) (right). Adapted from ref [14]. (d) Schematic view of α₁1.2 subunit harboring the T1066Y mutation at IIIS6 that renders the channel Nif-resistant, and the L775P mutation at IIS6 that renders the channel Ca²⁺ impermeable. (e) Macroscopic whole-cell Ca²⁺ currents (I_Ca) elicited from a holding potential of −80 mV to various test potentials in response to a 200 ms test pulse in Xenopus oocytes expressing GFP-tagged α₁1.2 or GFP-tagged α₁1.2^L775P with α2δ/β2A (inset). Representative traces and leak-subtracted peak current-voltage relationships of wt Cav1.2 and impermeable channel. Data collected from oocytes (n = 12 − 15), expressing α₁1.2/α2δ/β2A (open circle), α₁1.2^L775P/α2δ/β2A (close circle), and α2δ/β2A subunits (rectangle). (f) Amperometry currents triggered by K60 from single bovine chromaffin cells infected with either Nif resistance wt α₁1.2 or Nif resistance impermeable α₁1.2^L775P mutant with 2 mM Ca²⁺ as the charge carrier, with or without Cav1.2 agonist FPL (0.5 μM) in the presence of 5 μM Nif. (g) Cumulative distribution of spikes plotted versus time after the onset of membrane depolarization in cells infected with wt α₁1.2 subunit (left panel) or mutated α₁1.2^L775P subunit (right panel) in the presence (●, ♦) and in the absence of 0.5 μm FPL (○, ◇), respectively. Inset, expanded scale of the cumulative events elicited by K60 from single cells infected with either wt α₁1.2 subunit (left panel) or impermeable α₁1.2/L775P (right panel) with 2 mM Ca²⁺ as the charge carrier, emphasizing initial rates (10–30 s) and sustained rates (30–60 s). Adapted from ref [63].

Catecholamine (CA) release, monitored by amperometry of single bovine chromaffin cells showed that a single-point Cav1.2 mutant rendering the channel Ca2+-impermeable, mediates membrane depolarization triggered CA release, similar to wt Cav1.2. Membrane depolarization also induces CA release when Ca2+ is substituted with the impermeable cation La3+. Hence, evoked release mediated by a Ca2+ impermeable channel or by ion-pore impermeable cation suggest that ES coupling is critically dependent on ion pore occupancy rather than calcium entry. These results substantiate a noninotropic conformational coupling activity of VGCC as the mechanism underlying ES coupling.

Figure 7. The non-ionotropic activity of Cav1.2 mediates excitation secretion (ES) coupling. A schematic illustration of the closed state of VGCCs in the closed state occupied by a single Ca²⁺ ion tightly bound (<1 µM) to the EEEE motif (left). Upon arrival of an action potential (xxxv), the open channel, now occupied with additional Ca²⁺ ions, triggered fast (µs) fusion of a functionally VGCC-associate primed vesicle, prior to ion flow (non-ionotropic activity) (middle). Ca²⁺ inflow (ionotropic activity) elevates [Ca²⁺]_i, which is essential for operating Ca²⁺-dependent intracellular activities, such as VGCC inactivation (CDI), and vesicle priming (right).

A schematic illustration of VGCC mediating ES coupling in two distinct steps. In the inactive closed state, a single tightly bound calcium ion occupies the selectivity filter within the channel. Upon the arrival of an action potential, conformational changes occur simultaneously with channel opening, facilitating the binding of additional calcium ions to the selectivity filter. These conformational changes trigger rapid (µs) transmitter release preceding the influx of Ca2+ in a non-ionotropic manner. The subsequent ionotropic step involves Ca2+ entry, characterized by Ca2+-dependent closure of the channels and other Ca2+-dependent intracellular processes such as vesicle priming.

Figure 8. Ca²⁺-impermeable channel Cav1.2^L745P mediates excitation contraction (EC) coupling in cardiomyocytes, and requires intact selectivity filter. (a) Cardiac excitation – contraction coupling triggered by electrical stimulation in the absence (left upper) and presence (right upper) of 8 μM Nif in control intact cardiomyocytes, in cells infected with the Nif-resistant functional α₁1.2^T1066Y subunit (left lower) and the Nif-resistant α₁1.2^L775P/T1066Y mutant, in the presence of 8 μM Nif (right lower). (b) Frequency of Ca²⁺ transients in the presence and absence of Nif of control cells, and cells infected with α₁1.2^T1066Y or α₁1.2^L775P/T1066Y. Data are shown as means ± SEM and analyzed by a Student’s t test. **p < 0.001 (n = 20). (c) Most of the cardiomyocytes infected with the Nif-resistant α₁1.2^T1066Y (90%) or Nif-resistant α₁1.2^L775P/T1066Y (94%), responded to cell stimulation in the presence of Nif, as compared with 12% of the control cells. (d), Schematic view of the α₁1.2 subunit of Cav1.2 harboring the T1066Y mutation at IIIS6, providing Nif-resistant, a L745P mutation at IIS6, providing Ca²⁺-impermeability, and quadruple mutations EEEE/AAAA preventing ion-pore occupancy. (e) Representative 410 nm to 490 nm traces elicited in response to electric stimulation in control cells in the absence (top, left) and presence of 8 μM Nif (top right), α₁1.2^L775P/T1066Y infected cells (bottom left), or α₁1.2^{L775P/T1066Y/4A} infected cells (bottom right) in the presence of 8 μM Nif. (f) Frequency of depolarization-evoked Ca²⁺ transients in control and infected cells. Data is shown as means ± SEM and analyzed by a Student’s t test. **p < 0.001 (n = 20). Adapted from ref [21].

Membrane depolarization of neonate cardiomyocytes infected with a lentivirus encoded by a nifedipine (Nif)-resistant Ca2+-impermeable Cav1.2-induced cardiomyocyte contraction, assessed using the fluorescence ratio of the Ca2+-sensitive dye Indo-1. This is consistent with non-ionotropic and direct Cav1.2/RyR2 signaling. The Ca2+-impermeable Cav1.2 channel was further mutated, replacing the four Glu comprising the selectivity filter with Ala. This mutant failed to elicited contraction, suggesting that EC coupling in the neonate cardiomyocytes requires extracellular Ca2+ for occupancy of the ion pore rather than intracellular Ca2+.

Figure 9. The non-ionotropic activity of Cav1.2 mediates excitation contraction (EC) coupling in cardiac cells. A schematic illustration of the closed state of VGCCs occupied by a single Ca²⁺ ion tightly bound (<1 µM) to the EEEE motif (left). Upon arrival of an action potential (xxxv), the open channel, now occupied with an additional Ca²⁺ ion(s), triggers cardiac contraction most likely through a direct interaction with RyR2 (an unidentified site), prior to ion flow (non-ionotropic activity) (middle). Ca²⁺ inflow (ionotropic activity) elevates [Ca²⁺]_i, which is essential for Ca²⁺-dependent intracellular activities, for example, replenishing SR stores, Cav1.2 inactivation (right).

A schematic illustration of VGCC mediating EC coupling in cardiac neonate cardiomyocytes in two distinct steps. In the inactive closed state, a single tightly bound calcium ion occupies the selectivity filter within the channel. Upon the arrival of an action potential, conformational changes occur simultaneously with channel opening, facilitating the binding of additional calcium ions to the selectivity filter. These conformational changes trigger cardiac contraction preceding the influx of Ca2+ in a non-ionotropic manner. The subsequent ionotropic step involves Ca2+ entry, characterized by Ca2+-dependent closure of the channels and other Ca2+-dependent intracellular processes such as NCX-1 activation.

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Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.