Cardiac-type EC-coupling in dysgenic myotubes restored with Ca2+ channel subunit isoforms alpha1C and alpha1D does not correlate with current density

Ca(2+)-induced Ca(2+)-release (CICR)-the mechanism of cardiac excitation-contraction (EC) coupling-also contributes to skeletal muscle contraction; however, its properties are still poorly understood. CICR in skeletal muscle can be induced independently of direct, calcium-independent activation of sarcoplasmic reticulum Ca(2+) release, by reconstituting dysgenic myotubes with the cardiac Ca(2+) channel alpha(1C) (Ca(V)1.2) subunit. Ca(2+) influx through alpha(1C) provides the trigger for opening the sarcoplasmic reticulum Ca(2+) release channels. Here we show that also the Ca(2+) channel alpha(1D) isoform (Ca(V)1.3) can restore cardiac-type EC-coupling. GFP-alpha(1D) expressed in dysgenic myotubes is correctly targeted into the triad junctions and generates action potential-induced Ca(2+) transients with the same efficiency as GFP-alpha(1C) despite threefold smaller Ca(2+) currents. In contrast, GFP-alpha(1A), which generates large currents but is not targeted into triads, rarely restores action potential-induced Ca(2+) transients. Thus, cardiac-type EC-coupling in skeletal myotubes depends primarily on the correct targeting of the voltage-gated Ca(2+) channels and less on their current size. Combined patch-clamp/fluo-4 Ca(2+) recordings revealed that the induction of Ca(2+) transients and their maximal amplitudes are independent of the different current densities of GFP-alpha(1C) and GFP-alpha(1D). These properties of cardiac-type EC-coupling in dysgenic myotubes are consistent with a CICR mechanism under the control of local Ca(2+) gradients in the triad junctions.     

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle.     

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.     

Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle

Background

The multisubunit (α_1S,α₂-δ, β_1a and γ₁) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca²⁺ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca²⁺ channel. To more fully investigate the function of the γ₁ subunit in these two processes, we produced mice lacking this subunit by gene targeting.

Results

Mice lacking the DHPR γ₁ subunit (γ₁ null) survive to adulthood, are fertile and have no obvious gross phenotypic abnormalities. The γ₁ subunit is expressed at approximately half the normal level in heterozygous mice (γ₁ het). The density of the L-type Ca²⁺ current in γ₁ null and γ₁ het myotubes was higher than in controls. Inactivation of the Ca²⁺ current produced by a long depolarization was slower and incomplete in γ₁ null and γ₁ het myotubes, and was shifted to a more positive potential than in controls. However, the half-activation potential of intramembrane charge movements was not shifted, and the maximum density of the total charge was unchanged. Also, no shift was observed in the voltage-dependence of Ca²⁺ transients. γ₁ null and γ₁ het myotubes had the same peak Ca²⁺ amplitude vs. voltage relationship as control myotubes.

Conclusions

The L-type Ca²⁺ channel function, but not the SR Ca²⁺ release triggering function of the skeletal muscle dihydropyridine receptor, is modulated by the γ₁ subunit.     

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.     

Biochemical correlates of alpha 1-adrenoceptor activation.

An impressive body of evidence based on radioligand binding, biochemical, functional and structural data supports the existence of two sub-types of the alpha 1-adrenoceptor (alpha 1a and alpha 1b) in the CNS and in peripheral tissues. Both elevate intracellular Ca2+ concentrations, although by different mechanisms, resulting in a cascade of intracellular events dependent upon the cell type involved.     

Recovery of Ca2+ current, charge movements, and Ca2+ transients in myotubes deficient in dihydropyridine receptor beta 1 subunit transfected with beta 1 cDNA.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients of mouse dihydropyridine receptor (DHPR) beta 1-null myotubes expressing a mouse DHPR beta 1 cDNA have been characterized. In beta 1-null myotubes maintained in culture for 10-15 days, the density of the L-type current was approximately 7-fold lower than in normal cells of the same age (Imax was 0.65 +/- 0.05 pA/pF in mutant versus 4.5 +/- 0.8 pA/pF in normal), activation of the L-type current was significantly faster (tau activation at +40 mV was 28 +/- 7 ms in mutant versus 57 +/- 8 ms in normal), charge movements were approximately 2.5-fold lower (Qmax was 2.5 +/- 0.2 nC/microF in mutant versus 6.3 +/- 0.7 nC/microF in normal), Ca2+ transients were not elicited by depolarization, and spontaneous or evoked contractions were absent. Transfection of beta 1-null cells by lipofection with beta 1 cDNA reestablished spontaneous or evoked contractions in approximately 10% of cells after 6 days and approximately 30% of cells after 13 days. In contracting beta 1-transfected myotubes there was a complete recovery of the L-type current density (Imax was 4 +/- 0.9 pA/pF), the kinetics of activation (tau activation at +40 mV was 64 +/- 5 ms), the magnitude of charge movements (Qmax was 6.7 +/- 0.4 nC/microF), and the amplitude and voltage dependence of Ca2+ transients evoked by depolarizations. Ca2+ transients of transfected cells were unaltered by the removal of external Ca2+ or by the block of the L-type Ca2+ current, demonstrating that a skeletal-type excitation-contraction coupling was restored. The recovery of the normal skeletal muscle phenotype in beta 1-transfected beta-null myotubes shows that the beta 1 subunit is essential for the functional expression of the DHPR complex.     

Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.     

Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit.

The Ca2+ currents, charge movements, and intracellular Ca2+ transients in mouse skeletal muscle cells homozygous for a null mutation in the cchb1 gene encoding the beta 1 subunit of the dihydropyridine receptor have been characterized. I beta null, the L-type Ca2+ current of mutant cells, had a approximately 13-fold lower density than the L-type current of normal cells (0.41 +/- 0.042 pA/pF at + 20 mV, compared with 5.2 +/- 0.38 pA/pF in normal cells). I beta null was sensitive to dihydropyridines and had faster kinetics of activation and slower kinetics of inactivation than the L-type current of normal cells. Charge movement was reduced approximately 2.8-fold, with Qmax = 6.9 +/- 0.61 and Qmax = 2.5 +/- 0.2 nC/microF in normal and mutant cells, respectively. Approximately 40% of Qmax was nifedipine sensitive in both groups. In contrast to normal cells, Ca2+ transients could not be detected in mutant cells at any test potential; however, caffeine induced a robust Ca2+ transient. In homogenates of mutant muscle, the maximum density of [3H]PN200-110 binding sites (Bmax) was reduced approximately 3.9-fold. The results suggest that the excitation-contraction uncoupling of beta 1-null skeletal muscle involves a failure of the transduction mechanism that is due to either a reduced amount of alpha 1S subunits in the membrane or the specific absence of beta 1 from the voltage-sensor complex.     

Altered inactivation of Ca2+ current and Ca2+ release in mouse muscle fibers deficient in the DHP receptor gamma1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit gamma1 have previously been studied using coexpression with the cardiac alpha1C polypeptide in nonmuscle cells and primary-cultured myotubes of gamma1-deficient mice. Data from single adult muscle fibers of gamma-/- mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from gamma+/+ and gamma-/- mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of -80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage-dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in gamma-/- compared with gamma+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the gamma1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse.     

Mutations in the alpha1 subunit of an L-type voltage-activated Ca2+ channel cause myotonia in Caenorhabditis elegans.

The control of excitable cell action potentials is central to animal behavior. We show that the egl-19 gene plays a pivotal role in regulating muscle excitation and contraction in the nematode Caenorhabditis elegans and encodes the alphal subunit of a homologue of vertebrate L-type voltage-activated Ca2+ channels. Semi-dominant, gain-of-function mutations in egl-19 cause myotonia: mutant muscle action potentials are prolonged and the relaxation delayed. Partial loss-of-function mutations cause slow muscle depolarization and feeble contraction. The most severe loss-of-function mutants lack muscle contraction and die as embryos. We localized two myotonic mutations in the sixth membrane-spanning domain of the first repeat (IS6) region, which has been shown to be responsible for voltage-dependent inactivation. A third myotonic mutation implicates IIIS4, a region involved in sensing plasma-membrane voltage change, in the inactivation process.     

Store-operated Ca2+ current in prostate cancer epithelial cells. Role of endogenous Ca2+ transporter type 1

Ca(2+) influx via store-operated channels (SOCs) following stimulation of the plasma membrane receptors is the key event controlling numerous processes in nonexcitable cells. The human transient receptor potential vanilloid type 6 channel, originally termed Ca(2+) transporter type 1 (CaT1) protein, is one of the promising candidates for the role of endogenous SOC, although investigations of its functions have generated considerable controversy. In order to assess the role of CaT1 in generating endogenous store-operated Ca(2+) current (I(SOC)) in the lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cell line, we manipulated its endogenous levels by means of antisense hybrid depletion or pharmacological up-regulation (antiandrogen treatment) combined with functional evaluation of I(SOC). Antisense hybrid depletion of CaT1 decreased I(SOC) in LNCaP cells by approximately 50%, whereas enhancement of CaT1 levels by 60% in response to Casodex treatment potentiated I(SOC) by 30%. The functional characteristics of I(SOC) in LNCaP cells were similar in many respects to those reported for heterologously expressed CaT1, although 2-aminoethoxydiphenyl borate sensitivity and lack of constitutive current highlighted notable departures. Our results suggest that CaT1 is definitely involved in I(SOC), but it may constitute only a part of the endogenous SOC, which in general may be a heteromultimeric channel composed of homologous CaT1 and other transient receptor potential subunits.     

Latency correlates with period in a model for signal-induced Ca2+ oscillations based on Ca2(+)-induced Ca2+ release.

Oscillations in cytosolic Ca2+ develop in a variety of cells after an induction phase, called latency, the duration of which depends on the magnitude of external stimulation. Experiments in hepatocytes indicate that the period and latency of Ca2+ oscillations both decrease as the level of the stimulus increases. We analyze the correlation between period and latency in a model recently proposed for signal-induced Ca2+ oscillations. We show that the linear relationship between period and latency observed in the experiments arises naturally in this model as a result of the mechanism of Ca2(+)-induced Ca2+ release on which it is based.     

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.     

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.     

The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit

The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.     

Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation

STIM1 and Orai1 are essential components of Ca(2+) release-activated Ca(2+) channels (CRACs). After endoplasmic reticulum Ca(2+) store depletion, STIM1 in the endoplasmic reticulum aggregates and migrates toward the cell periphery to co-localize with Orai1 on the opposing plasma membrane. Little is known about the roles of different domains of STIM1 and Orai1 in protein clustering, migration, interaction, and, ultimately, opening CRAC channels. Here we demonstrate that the coiled-coil domain in the C terminus of STIM1 is crucial for its aggregation. Amino acids 425-671 of STIM1, which contain a serine-proline-rich region, are important for the correct targeting of the STIM1 cluster to the cell periphery after calcium store depletion. The polycationic region in the C-terminal tail of STIM1 also helps STIM1 targeting but is not essential for CRAC channel activation. The cytoplasmic C terminus but not the N terminus of Orai1 is required for its interaction with STIM1. We further identify a highly conserved region in the N terminus of Orai1 (amino acids 74-90) that is necessary for CRAC channel opening. Finally, we show that the transmembrane domain of Orai1 participates in Orai1-Orai1 interactions.     

Ca2+-calmodulin-dependent protein kinase II potentiates store-operated Ca2+ current

A rise in intracellular Ca2+ (Ca2+i) mediates various cellular functions ranging from fertilization to gene expression. A ubiquitous Ca2+ influx pathway that contributes significantly to the generation of Ca2+i signals, especially in non-excitable cells, is store-operated Ca2+ entry (SOCE). Consequently, the modulation of SOCE current affects Ca2+i dynamics and thus the ensuing cellular response. Therefore, it is important to define the mechanisms that regulate SOCE. Here we show that a rise in Ca2+i potentiates SOCE. This potentiation is mediated by Ca2+-calmodulin-dependent protein kinase II (CaMKII), because inhibition of endogenous CaMKII activity abrogates Ca2+i-mediated SOCE potentiation and expression of constitutively active CaMKII potentiates SOCE current independently of Ca2+i. Moreover, we present evidence that CaMKII potentiates SOCE by altering SOCE channel gating. The regulation of SOCE by CaMKII defines a novel modulatory mechanism of SOCE with important physiological consequences.