The inhibition effect of uncarialin A on voltage-dependent L-type calcium channel subunit alpha-1C: Inhibition potential and molecular stimulation.

Cardiovascular diseases, such as hypertension and cardiac failure, have become the most major and global cause for threatening human health in recent years. Uncaria rhynchophylla as a traditional Chinese medicine is widely used to treat hypertension for a long history, whereas its medicinal effective components and potential action mechanism are uncertain. Therefore, twenty-four alkaloids (1-24) isolated from U. rhynchophylla were assayed for their relaxant effects against phenylephrine (Phe)-induced contraction of rat mesenteric arteries. Among them, we surprisingly found that uncarialin A (21) exhibited most potent relaxation effect against Phe-induced contraction (IC50 = 0.18 µM) in the manner of independent on endothelium-derived vasorelaxing factors and endothelium. All the experiments including measurement of Ca2+ in vascular smooth muscle cells (VSMCs) by fluorescence microscopy, whole-cell path clamp, molecular docking, and molecular dynamics, demonstrated that uncarialin A (21) could significantly inhibit L-type calcium channel subunit alpha-1C (Cav1.2) via the hydrogen bond interaction with amino acid residue Met1186, allowing the inhibition of Ca2+ inward current. Our results suggested that uncarialin A (21) could be served as a potential L-type Cav1.2 blocker in the effective treatment of cardiovascular diseases.

Calcium Supplementation Enhanced Adipogenesis and Improved Glucose Homeostasis Through Activation of Camkii and PI3K/Akt Signaling Pathway in Porcine Bone Marrow Mesenchymal Stem Cells (pBMSCs) and Mice Fed High Fat Diet (HFD).

BACKGROUND/AIMS: It has been implicated that calcium supplementation is involved in reducing body weight/fat and improving glucose homeostasis. However, the underlying mechanisms are still not fully understood. Here, we investigated the effects of calcium supplementation on adipogenesis and glucose homeostasis in porcine bone marrow mesenchymal stem cells (pBMSCs) and high fat diet (HFD)-fed mice and explored the involved signaling pathways. METHODS: In vitro, pBMSCs were treated with 4 mM extracellular calcium ([Ca2+]o) and/or 1 µM nifedipine, 0.1 µM BAPTA-AM, 1 µM KN-93, 50 nM wortmannin for 10 days. The intracellular calcium ([Ca2+]i) levels were measured using Fluo 3-AM by flow cytometry. The adipogenic differentiation of pBMSCs was determined by Oil Red-O staining and triglyceride assay. The expression of marker genes involved in adipogenesis (peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα)) and glucose uptake (glucose transporter 4 (GLUT4)), as well as the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and PI3K/Akt-FoxO1/AS160 signaling pathways were determined by Western blotting. Glucose uptake and utilization were examined using 2-NBDG assay and glucose content assay, respectively. In vivo, C57BL/6J male mice were fed a HFD (containing 1.2% calcium) without or with 0.6% (w/w) calcium chloride in drinking water for 13 weeks. The adipogenesis, glucose homeostasis and the involvement of CaMKII and PI3K/Akt signaling pathway were also assessed. RESULTS: In vitro, [Ca2+]o stimulated pBMSCs adipogenesis by increasing [Ca2+]i level and activating CaMKII and PI3K/Akt-FoxO1 pathways. In addition, [Ca2+]o promoted glucose uptake/utilization by enhancing AS160 phosphorylation, GLUT4 expression and translocation. However, the stimulating effects of [Ca2+]o on pBMSCs adipogenesis and glucose uptake/utilization were abolished by L-VGCC blocker Nifedipine, [Ca2+]i chelator BAPTA-AM, CaMKII inhibitor KN-93, or PI3K inhibitor Wortmannin. In vivo, calcium supplementation decreased body weight and fat content, increased adipocyte number, and improved glucose homeostasis, with elevated PPARγ and GLUT4 expression and PI3K/Akt activation in iWAT. CONCLUSION: calcium supplementation enhanced adipogenesis and glucose uptake in pBMSCs, which was coincident with the increased adipocyte number and improved glucose homeostasis in HFD-fed mice, and was associated with activation of CaMKII and PI3K/Akt-FoxO1/AS160 pathways. These data provided a broader understanding of the mechanisms underlying calcium-induced body weight/fat loss and glycemic control.

Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx.

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 µM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Antagonism of L-type Ca(v) channels with nifedipine differentially affects performance of wildtype and NK1R-/- mice in the 5-Choice Serial Reaction-Time Task.

Mice with functional ablation of the substance P-preferring receptor gene ('Nk1r' in mice ('NK1R-/-'), 'TACR1' in humans) display deficits in cognitive performance that resemble those seen in patients with Attention Deficit Hyperactivity Disorder (ADHD): namely, inattentiveness, impulsivity and perseveration. A recent report suggested that the L-type Ca(v) channel blocker, nifedipine, can ameliorate behavioral abnormalities of this type in humans. In light of evidence that NK1R antagonists modulate the opening of these L-type channels, we investigated whether nifedipine modifies %premature responses (impulsivity), perseveration or %omissions (inattentiveness) in the 5-Choice Serial Reaction-Time Task (5-CSRTT) and whether the response differs in NK1R-/- and wildtype mice. %Premature responses and perseveration were reduced in both genotypes, although wildtype mice were more sensitive to the effects of nifedipine than NK1R-/- mice. By contrast, nifedipine greatly increased %omissions but, again, was more potent in wildtypes. %Accuracy and locomotor activity were unaffected in either genotype. We infer that behavior of mice in the 5-CSRTT depends on the regulation of striato-cortical networks by L-type Ca(v) channels and NK1R. We further suggest that disruption of NK1R signaling in patients with ADHD, especially those with polymorphisms of the TACR1 gene, could lead to compensatory changes in the activity of L-type channels that underlie or exacerbate their problems. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Modulation of calcium-dependent inactivation of L-type Ca2+ channels via ß-adrenergic signaling in thalamocortical relay neurons.

Neuronal high-voltage-activated (HVA) Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we have shown that ß-adrenergic receptor (ßAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14-22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after ßAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via ßAR stimulation requires a protein complex consisting of Ca(V)1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca(2+) channels allowing their phosphorylation and thereby modulation of CDI.

Tissue slices from adult mammalian hearts as a model for pharmacological drug testing.

AIM: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. METHOD: Vibratome cut ventricular slices (350 microm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). RESULTS: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD(90), 169.9 +/- 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD(90) with a half maximal effective concentration (EC(50)) of 4.5 microM. The I(Kr) blocker E4031 and neuroleptic drug risperidone prolonged APD(90) with EC(50) values of 31 nM and 0.67 microM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m s(-1). CONCLUSION: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research.

Different effects of L/N-type and L-type calcium channel blockers on the renin-angiotensin-aldosterone system in SHR/Izm.

BACKGROUND: The L/N-type calcium channel blocker (CCB) cilnidipine has been demonstrated to suppress sympathetic nerve activity. In the present study, we investigated the effects of cilnidipine on the renin-angiotensin-aldosterone system (RAAS) in SHR/Izm rats to confirm differences from the effects of L-type CCB. METHODS: Male SHR/Izm rats received vehicle, cilnidipine (0.3, 3 mg/kg) or amlodipine (0.3, 3 mg/kg) by gavage for systolic blood pressure (SBP) measurement. For biochemical analyses, the experiments were performed under anesthesia. RESULTS: Low doses of cilnidipine or amlodipine had no effect on SBP or RAAS parameters. A high dose of either drug produced similar effects on SBP levels. Although cilnidipine had no effect on plasma renin activity or the plasma angiotensin II level, amlodipine significantly increased these parameters as compared to levels in the vehicle group. The cilnidipine group had a significantly lower plasma aldosterone level and renal cortical tissue norepinephrine level than the vehicle group. CONCLUSIONS: Cilnidipine had effects different from those of L-type CCB on the RAAS in SHR/Izm rats. Our results indicate that suppression of RAAS hyperactivity by cilnidipine can be partly explained by its sympatholytic action.

A genetic screen for dihydropyridine (DHP)-resistant worms reveals new residues required for DHP-blockage of mammalian calcium channels.

Dihydropyridines (DHPs) are L-type calcium channel (Ca(v)1) blockers prescribed to treat several diseases including hypertension. Ca(v)1 channels normally exist in three states: a resting closed state, an open state that is triggered by membrane depolarization, followed by a non-conducting inactivated state that is triggered by the influx of calcium ions, and a rapid change in voltage. DHP binding is thought to alter the conformation of the channel, possibly by engaging a mechanism similar to voltage dependent inactivation, and locking a calcium ion in the pore, thereby blocking channel conductance. As a Ca(v)1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Ca(v)1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Ca(v)1 residues important for DHP-sensitivity, we screened 440,000 randomly mutated Caenorhabditis elegans genomes for worms resistant to DHP-induced growth defects. We identified 30 missense mutations in the worm Ca(v)1 pore-forming (alpha(1)) subunit, including eleven in conserved residues known to be necessary for DHP-binding. The remaining polymorphisms are in eight conserved residues not previously associated with DHP-sensitivity. Intriguingly, all of the worm mutants that we analyzed phenotypically exhibited increased channel activity. We also created orthologous mutations in the rat alpha(1C) subunit and examined the DHP-block of current through the mutant channels in culture. Six of the seven mutant channels examined either decreased the DHP-sensitivity of the channel and/or exhibited significant residual current at DHP concentrations sufficient to block wild-type channels. Our results further support the idea that DHP-block is intimately associated with voltage dependent inactivation and underscores the utility of C. elegans as a screening tool to identify residues important for DHP interaction with mammalian Ca(v)1 channels.

Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaValpha1 AID-CaVbeta interaction site that is critical for channel modulation.

Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaValpha1) and cytoplasmic (CaVbeta) subunits associate through a high-affinity interaction between the CaValpha1 alpha interaction domain (AID) and CaVbeta alpha binding pocket (ABP). Here we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry. We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVbeta with similar nanomolar affinities. Although the AID-ABP interface encompasses 24 side chains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVbeta. Together, the data support the primacy of the AID-ABP interface for CaValpha1-CaVbeta association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaValpha1-CaVbeta complex formation.

Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle.

In skeletal muscle, dihydropyridine receptors (DHPRs) in the plasma membrane interact with the type 1 ryanodine receptor (RyR1) at junctions with the sarcoplasmic reticulum. This interaction organizes junctional DHPRs into groups of four termed tetrads. In addition to the principle alpha1S subunit, the beta1a subunit of the DHPR is also important for the interaction with RyR1. To probe this interaction, we measured fluorescence resonance energy transfer (FRET) of beta1a subunits labeled with cyan fluorescent protein (CFP) and/or yellow fluorescent protein (YFP). Expressed in dysgenic (alpha1S-null) myotubes, YFP-beta1a-CFP and CFP-beta1a-YFP were diffusely distributed in the cytoplasm and highly mobile as indicated by fluorescence recovery after photobleaching. Thus, beta1a does not appear to bind to other cellular proteins in the absence of alpha1S. FRET efficiencies for these cytoplasmic beta1a subunits were approximately 6-7%, consistent with the idea that <10 nm separates the N and C termini. After coexpression with unlabeled alpha1S (in dysgenic or beta1-null myotubes), both constructs produced discrete fluorescent puncta, which correspond to assembled DHPRs in junctions and that did not recover after photobleaching. In beta1-null myotubes, FRET efficiencies of doubly labeled beta1a in puncta were similar to those of the same constructs diffusely distributed in the cytoplasm and appeared to arise intramolecularly, since no FRET was measured when mixtures of singly labeled beta1a (CFP or YFP at the N or C terminus) were expressed in beta1-null myotubes. Thus, DHPRs in tetrads may be arranged such that the N and C termini of adjacent beta1a subunits are located >10 nm from one another.

Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling.

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR_ _R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR_ _R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.

Cav1.4 encodes a calcium channel with low open probability and unitary conductance.

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.

Amino acid substitutions in the pore of the Ca(V)1.2 calcium channel reduce barium currents without affecting calcium currents.

Ba(2+) currents through Ca(V)1.2 Ca(2+) channels are typically twice as large as Ca(2+) currents. Replacing Phe-1144 in the pore-loop of domain III with glycine and lysine, and Tyr-1152 with lysine, reduces whole-cell G(Ba)/G(Ca) from 2.2 (wild-type) to 0.95, 1.21, and 0.90, respectively. Whole-cell and single-channel measurements indicate that reductions in G(Ba)/G(Ca) result specifically from a decrease in Ba(2+) conductance and not changes in V(h) or P(O). Half-maximal block of I(Li) is increased by 3.2-, 3.8-, and 1.6-fold in Ca(2+), and 3.8-, 4.2-, and 1.8-fold in Ba(2+) for F1144G, Y1152K, and F1144K, respectively. High affinity interactions of individual divalent cations to the pore are not important for determining G(Ba)/G(Ca), because the fold increases in IC(50) values for Ba(2+) and Ca(2+) are similar. On the contrary, conductance-concentration curves indicate that G(Ba)/G(Ca) is reduced because the interactions of multiple Ba(2+) ions in the mutant pores are altered. The complexity of these interactions is exemplified by the anomalous mole fraction effect, which is flattened for F1144G and FY/GK but accentuated for F1144K. In summary, the physicochemical properties of the amino acid residues at positions 1144 and 1152 are crucial to the pore's ability to distinguish between multiple Ba(2+) ions and Ca(2+) ions.

Multiple loops of the dihydropyridine receptor pore subunit are required for full-scale excitation-contraction coupling in skeletal muscle.

Understanding which cytosolic domains of the dihydropyridine receptor participate in excitation-contraction (EC) coupling is critical to validate current structural models. Here we quantified the contribution to skeletal-type EC coupling of the alpha1S (CaV1.1) II-III loop when alone or in combination with the rest of the cytosolic domains of alpha1S. Chimeras consisting of alpha1C (CaV1.2) with alpha1S substitutions at each of the interrepeat loops (I-II, II-III, and III-IV loops) and N- and C-terminal domains were evaluated in dysgenic (alpha1S-null) myotubes for phenotypic expression of skeletal-type EC coupling. Myotubes were voltage-clamped, and Ca2+ transients were measured by confocal line-scan imaging of fluo-4 fluorescence. In agreement with previous results, the alpha1C/alpha1S II-III loop chimera, but none of the other single-loop chimeras, recovered a sigmoidal fluorescence-voltage curve indicative of skeletal-type EC coupling. To quantify Ca2+ transients in the absence of inward Ca2+ current, but without changing the external solution, a mutation, E736K, was introduced into the P-loop of repeat II of alpha1C. The Ca2+ transients expressed by the alpha1C(E736K)/alpha1S II-III loop chimera were approximately 70% smaller than those expressed by the Ca2+-conducting alpha1C/alpha1S II-III variant. The low skeletal-type EC coupling expressed by the alpha1C/alpha1S II-III loop chimera was confirmed in the Ca2+-conducting alpha1C/alpha1S II-III loop variant using Cd2+ (10(-4) M) as the Ca2+ current blocker. In contrast to the behavior of the II-III loop chimera, Ca2+ transients expressed by an alpha1C/alpha1S chimera carrying all tested skeletal alpha1S domains (all alpha1S interrepeat loops, N- and C-terminus) were similar in shape and amplitude to wild-type alpha1S, and did not change in the presence of the E736K mutation or in the presence of 10(-4) M Cd2+. Controls indicated that similar dihydropyridine receptor charge movements were expressed by the non-Ca2+ permeant alpha1S(E1014K) variant, the alpha1C(E736K)/alpha1S II-III loop chimera, and the alpha1C(E736K)/alpha1S chimera carrying all tested alpha1S domains. The data indicate that the functional recovery produced by the alpha1S II-III loop is incomplete and that multiple cytosolic domains of alpha1S are necessary for a quantitative recovery of the EC-coupling phenotype of skeletal myotubes. Thus, despite the importance of the II-III loop there may be other critical determinants in alpha1S that influence the efficiency of EC coupling.

Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels.

Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.

Calmodulin mediates Ca2+ sensitivity of sodium channels.

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca(2+)-dependent conformational change seen in the Cav1.2.CaM complex. Furthermore, CaM binding to Nav1 C termini lowered Ca2+ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nav1 channels. The R1902C autism mutation conferred a Ca(2+)-dependent conformational change in Nav1.2 C terminus.CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function.

Modulation of cardiac Ca2+ channel by Gq-activating neurotransmitters reconstituted in Xenopus oocytes.

L-type dihydropyridine-sensitive voltage dependent Ca(2+) channels (L-VDCCs; alpha(1C)) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, was G(q)- and PLC-independent and Ca(2+)-dependent, but not Ca(2+)-mediated, as only basal levels of Ca(2+) were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations.

Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module.

Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 +/- 0.12 to 0.83 +/- 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 +/- 0.05 to 2.60 +/- 0.16 mM, and 1.15 +/- 0.03 to 2.11 +/- 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.

Ca2+-dependent excitation-contraction coupling triggered by the heterologous cardiac/brain DHPR beta2a-subunit in skeletal myotubes.

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) alpha1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR beta1a-subunit. It is unknown whether EC coupling domains present in the beta-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit alpha1S is paired with the heterologous heart/brain DHPR beta2a-subunit. Studies were conducted in primary cultured myotubes from beta1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The beta2a-mediated Ca2+ current recovered in beta1 KO myotubes lacking the endogenous DHPR beta1a-subunit verified formation of the alpha1S/beta1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely beta1 KO and RyR1 KO, beta2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage-dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of beta2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the beta2a-subunit. In beta1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by beta2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing beta1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca2+ current; 3), the voltage dependence of the Ca2+ transient was bell-shaped and the maximum was centered at approximately +30 mV, consistent with the voltage dependence of the Ca2+ current; and 4), Ca2+ currents and Ca2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by beta2a was inferred by the drastic reduction in maximal Ca2+ fluorescence at large positive potentials (DeltaF/Fmax) in double dysgenic/beta1 KO myotubes overexpressing the pore mutant alpha1S (E1014K) and beta2a. The data indicate that beta2a, upon interaction with the skeletal pore subunit alpha1S, overrides critical EC coupling determinants present in alpha1S. We propose that the alpha1S/beta pair, and not the alpha1S-subunit alone, controls the EC coupling signal in skeletal muscle.