Short Communication: Genetic Ablation of L-Type Ca2+ Channels Abolishes Depolarization-Induced Ca2+ Release in Arterial Smooth Muscle
Rationale: In arterial myocytes, membrane depolarization-induced Ca2+ release (DICR) from the sarcoplasmic reticulum (SR) occurs through a metabotropic pathway that leads to inositol trisphosphate synthesis independently of extracellular Ca2+ influx. Despite the fundamental functional relevance of DICR, its molecular bases are not well known.
Objective: Biophysical and pharmacological data have suggested that L-type Ca2+ channels could be the sensors coupling membrane depolarization to SR Ca2+ release. This hypothesis was tested using smooth muscle–selective conditional Cav1.2 knockout mice.
Methods and Results: In aortic myocytes, the decrease of Ca2+ channel density was paralleled by the disappearance of SR Ca2+ release induced by either depolarization or Ca2+ channel agonists. Cav1.2 channel deficiency resulted in almost abolition of arterial ring contraction evoked by DICR. Ca2+ channel–null cells showed unaltered caffeine-induced Ca2+ release and contraction.
Conclusion: These data suggest that Cav1.2 channels are indeed voltage sensors coupled to the metabolic cascade, leading to SR Ca2+ release. These findings support a novel, ion-independent, functional role of L-type Ca2+ channels linked to intracellular signaling pathways in vascular myocytes.
The rise of cytosolic Ca2+ concentration required for vascular smooth muscle (VSM) contraction is classically considered to be attributable to either transmembrane Ca2+ influx through voltage- and/or receptor-operated channels or to Ca2+ release from the sarcoplasmic reticulum (SR). However, in recent years a new SR Ca2+ release mechanism, depolarization-induced Ca2+ release (DICR), has been observed.1–4 DICR refers to Ca2+ release from the SR in the absence of any transmembrane Ca2+ influx and takes place by means of a metabotropic pathway that involves G protein/phospholipase C activation and subsequent synthesis of inositol 1,4,5-trisphosphate.5–7
Despite the fundamental biological and pathophysiological implications of DICR,7–10 its underlying molecular mechanisms are poorly known. Some G protein–coupled receptors (GPCRs) have been proposed to have intrinsic voltage sensitivity and act as “voltage sensors” for DICR.7,11 However the intrinsic voltage-sensitivity of GPCRs cannot fully account for DICR because it is observed in the absence of GPCR ligands and is inhibited by organic Ca2+ channel blockers.3 Moreover, Ca2+ release can be triggered by Ca2+ channel agonists in the absence of membrane potential changes, thus suggesting that sensitivity of the GPCR or the phosphatidylinositol cascade to depolarization is not the primary cause of DICR.3,8 We have proposed that voltage-gated Ca2+ channels are the sensors for DICR and coined the term “calcium channel–induced Ca2+ release” (CCICR) for this novel form of plasma membrane-SR interaction in VSM.3,12
A critical experimental test necessary for establishing the actual involvement of Ca2+ channels in coupling membrane potential to SR Ca2+ release requires the study of viable VSM cells lacking Ca2+ channels. Herein, we describe the changes to CCICR in a smooth muscle-specific and temporally-controlled L-type Cav1.2 Ca2+ channel knockout mouse model.13 Our data strongly support the view that Ca2+ channels are indeed voltage sensors essential for DICR in arterial smooth muscle.
Cell Dispersion and Patch Clamp Recordings
We used conditional Cav1.2 knockout mice.13 Aorta arteries were obtained from anesthetized adult animals (20 to 30 g). Macroscopic Ca2+, Na+ and K+ currents were recorded from dispersed patch clamped myocytes.3,8
Measurement of Cytosolic [Ca2+] and Contractility in Arterial Rings
Data are expressed as means±SE with the number (n) of experiments (single cells) or animals (arterial rings) indicated. Statistical analysis was performed by unpaired Student t test and one-way ANOVA. A value of P<0.05 was considered as statistically significant.
For additional methodological details, see the expanded Methods section in the Online Data Supplement, available at http://circres.ahajournals.org.
The electric parameters of the cells studied are summarized in Online Table I. Voltage-clamped aortic VSM cells showed depolarization activated inward currents with a large tail current at the end of the pulse (Figure 1A, top). These currents had the typical time course and voltage dependence (Figure 1B) of those mediated by voltage-gated Ca2+ channels in VSM cells.13,14 In Cav1.2+/− cells, the amplitude of the Ca2+ current decreased to approximately half the amplitude observed in control cells (Figure 1A, center; Online Table I), indicating lack of compensation by the functional allele. The Ca2+ currents almost disappeared in Cav1.2−/− cells (Figure 1A, bottom; Online Table I); however, the current/voltage relationship remained the same in all the cell classes (Figure 1B). In VSM cells, we recorded a transient Na+ current (Figure 1A, bottom; Online Table I) that disappeared after replacement of external Na+ with the impermeant cation N-methyl-d-glucamine (Figure 1C). Cav1.2-deficient cells showed a decrease in the amplitude of the voltage-dependent K+ current and variations in the passive electric parameters (Online Table I and Online Figure I).
To determine whether reduction in Ca2+ channel density influences CCICR, we performed experiments on dispersed Fura 2-loaded myocytes. Cell depolarization with 70 mmol/L external K+ (with 2.5 mmol/L Ca2+) induced a large rise of cytosolic [Ca2+] attributable to the influx of Ca2+ through Ca2+ channels (Figure 2A). After recovery, cells were bathed in a 0 Ca2+ plus 1 mmol/L EGTA solution to fully prevent any transmembrane Ca2+ influx and then exposed to high K+ plus FPL-64176, a Ca2+ channel agonist, to elicit CCICR.3,8 We used membrane depolarization and pharmacological Ca2+ channel activation to favor generation of CCICR signals even in cells with low density of Ca2+ channels.3 To evaluate normal cell responsiveness independent of Ca2+ channel activation, we applied caffeine to directly elicit Ca2+ release from ryanodine-sensitive SR stores.3
In Cav1.2-deficient animals (+/− and −/−), the high K+-induced rise of cytosolic [Ca2+] decreased in parallel with the reduction of Ca2+ current amplitude, although the response to caffeine remained unaltered or even increased (Figure 2B through 2D and Online Table I). Because the amplitude of 70K+-induced depolarization was ≈7 mV smaller in Cav1.2−/− than in wild-type myocytes (Online Table I and Online Figure II), we also tested in the Cav1.2-null cells the effect of 100K+ plus FPL solution. This last treatment evoked a depolarization shift of 36.8±5.4 mV (n=8), larger than that elicited by 70 K+ in control cells, which was also unable to trigger CCICR (Figure 2C and 2D and Online Figure II). CCICR triggered by high K+ in combination with BayK8644, another L-type Ca2+ channel agonist that potentiates CCICR,3 was also abolished in Cav1.2−/− myocytes (Online Figure III).
High-K+-evoked mechanical responses studied in aortic rings decreased in amplitude as did the changes in Ca2+ channel density and cytosolic [Ca2+]. In contrast, vasoconstriction induced by caffeine remained unaltered in the three arterial types studied (Figure 3A and Online Table I). Contraction dependent on CCICR (elicited in Ca2+-free solutions) (Figure 3B)8,10 progressively decreased in rings from Cav1.2+/− or Cav1.2−/− mice (Figure 3C).
In this report, we show that CCICR in VSM cells requires the presence of functional L-type Ca2+ channels. Using the Cav1.2 knockout mouse model,13 we have demonstrated that in the absence of extracellular Ca2+ (0 Ca2+ plus EGTA added) depolarization- or Ca2+ channel agonist–induced Ca2+ release and contraction decreased in parallel with reduction of Ca2+ channel density. Apart from the lack of Ca2+ currents, myocytes from knockout mice appeared to be healthy, because they had normal resting potential and expressed a transient voltage-dependent Na+ current similar in amplitude and kinetics to the Na+ current in wild-type cells. In addition, Cav1.2-null cells showed normal responses (rise of cytosolic [Ca2+] or contraction) to caffeine, a direct activator of SR Ca2+ release that bypasses activation of membrane channels or receptors. Input resistance, a highly sensitive indicator of membrane integrity, was even slightly increased in knockout cells.
The study of the CCICR signal in isolation requires the use of solutions without external Ca2+, a condition in which Na+ can permeate through Ca2+ channels. Thus, it could be argued that Na+ influx activates G protein–mediated Ca2+ release, because dissociation of G proteins by intracellular Na+ has been described in neurons.15 This possibility is unlikely because CCICR is seen in cells bathed in solutions without Na+ and after blockade of the Ca2+ channel pore with Cd2+ or Ni2+.3 Moreover, in the current experiments, CCICR was abolished in Cav1.2−/− cells despite most of them generated a large voltage-dependent Na+ current.
The current data, obtained after ablation of the Cav1.2 gene, support former pharmacological studies3,8 and suggest that voltage-dependent Ca2+ channels are, indeed, the voltage sensors for DICR in VSM cells. This is in fair agreement with the disappearance of the high K+-induced cytosolic Ca2+ waves in dysgenic skeletal muscle myotubes lacking the α1C subunit of the dihydropyridine receptor.6 Nevertheless, DICR is also observed in megakaryocytes that do not seem to express functional Ca2+ channels5 or in VSM cells that express the muscarinic receptor,4 which has intrinsic voltage dependence.11 Therefore, besides Ca2+ channels, other molecules might contribute to the coupling of membrane potential to SR Ca2+ release.
It has been reported that ion channels interact with modulatory binding partners to form dynamic intracellular signaling complexes and that membrane receptors, G protein, and L-type Ca2+ channels are in close proximity within membrane microdomains.16 Hence, it is conceivable that G protein activation might be mediated by interaction with the voltage-sensing α1 subunit of Cav1.2 channels. This concept (voltage-gated ion channel–mediated regulation of intracellular signaling pathways) is also compatible with data showing modulation of cell proliferation by K+ channels depending on the position of the voltage sensor by a mechanism that is independent of ion flux.17
Sources of Funding
Supported by grants from the Marcelino Botín Foundation, Junta de Andalucía, Ministry of Health (Fondo de Investigación Sanitaria, Ciberned and Recava), and Plan Nacional (Ministry of Science).
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Franco-Obregon A, Urena J, Lopez-Barneo J. Oxygen-sensitive calcium channels in vascular smooth muscle and their possible role in hypoxic arterial relaxation. Proc Natl Acad Sci U S A. 1995; 92: 4715–4719.
Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne MC, Hoshi T, Hell JW. A beta2 adrenergic receptor signaling complex assembled with the Ca2+ channel Cav1.2. Science. 2001; 293: 98–101.
Hegle AP, Marble DD, Wilson GF. A voltage-driven switch for ion-independent signaling by ether-a-go-go K+ channels. Proc Natl Acad Sci U S A. 2006; 103: 2886–2891.
Novelty and Significance
What Is Known?
Contraction of vascular smooth muscle depends on cytosolic [Ca2+].
The best-known mechanism for the rise in cytosolic [Ca2+] is the influx of the cation through Ca2+ channels of the plasma membrane.
What New Information Does This Article Contribute?
Ca2+ channels of arterial myocytes are voltage sensors that, in the absence of transmembrane Ca2+ influx, couple membrane depolarization to Ca2+ release from the sarcoplasmic reticulum.
This ion-independent function of Ca2+ channels explains how the cell membrane potential influences intracellular functions.
In vascular smooth muscle cells, membrane depolarization can induce Ca2+ release from the sarcoplasmic reticulum. We postulated that Ca2+ channels could be the voltage sensors coupling membrane potential to a G protein-dependent biochemical cascade leading to inositol trisphosphate synthesis and Ca2+ release from intracellular stores. This hypothesis was tested using genetically modified mice lacking the vascular smooth muscle-selective Ca2+ channel gene Cav1.2. In these smooth muscle cells, depolarization-induced Ca2+ release disappeared although other aspects of Ca2+ homeostasis remained intact. This report unveils a novel function of Ca2+ channels - linking membrane potential to intracellular signaling pathways. It also helps to explain the generation of pathological vasospasm in partially depolarized smooth muscle cells.
↵*Both authors have contributed equally to this work.
Original received November 28, 2009; revision received March 3, 2010; accepted March 4, 2010.