SNAP-Tag-Targeted MRI-Fluorescent Multimodal Probes.

Magnetic resonance imaging (MRI) is a powerful imaging modality, widely employed in research and clinical settings. However, MRI images suffer from low signals and a lack of target specificity. We aimed to develop a multimodal imaging probe to detect targeted cells by MRI and fluorescence microscopy. We synthesized a trifunctional imaging probe consisting of a SNAP-tag substrate for irreversible and specific labelling of cells, cyanine dyes for bright fluorescence, and a chelated GdIII molecule for enhancing MRI contrast. Our probes exhibit specific and efficient labelling of genetically defined cells (expressing SNAP-tag at their membrane), bright fluorescence and MRI signal. Our synthetic approach provides a versatile platform for the production of multimodal imaging probes, particularly for light microscopy and MRI.

Protein kinase A regulates C-terminally truncated CaV 1.2 in Xenopus oocytes: roles of N- and C-termini of the α1C subunit.

KEY POINTS: ß-Adrenergic stimulation enhances Ca2+ entry via L-type CaV 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV 1.2 remain controversial despite extensive research. We show that PKA regulation of CaV 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α1C , is truncated. The PKA upregulation of CaV 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the ß subunit of CaV 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV 1.2 in the heart. ABSTRACT: ß-Adrenergic stimulation enhances Ca2+ currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signalling via ß-adrenergic receptors (ß-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by ∼40% (rabbit α1C ) to ∼130% (mouse α1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaV ß2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α1C .

SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells.

Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the "voltage-clock," where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the "Ca(2+) clock," where cyclical release of Ca(2+) from Ca(2+) stores depolarizes the membrane during diastole via activation of the Na(+)-Ca(2+) exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called "voltage and Ca(2+) clock" pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the "voltage or Ca(2+) clock" produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca(2+)-activated intermediate K(+) conductance (IK(Ca), KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IK(Ca) inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IK(Ca) appears to play a crucial role in human embryonic cardiac automaticity.

Regulation of cardiac L-type Ca²âº channel CaV1.2 via the ß-adrenergic-cAMP-protein kinase A pathway: old dogmas, advances, and new uncertainties.

In the heart, adrenergic stimulation activates the ß-adrenergic receptors coupled to the heterotrimeric stimulatory Gs protein, followed by subsequent activation of adenylyl cyclase, elevation of cyclic AMP levels, and protein kinase A (PKA) activation. One of the main targets for PKA modulation is the cardiac L-type Ca²âº channel (CaV1.2) located in the plasma membrane and along the T-tubules, which mediates Ca²âº entry into cardiomyocytes. ß-Adrenergic receptor activation increases the Ca²âº current via CaV1.2 channels and is responsible for the positive ionotropic effect of adrenergic stimulation. Despite decades of research, the molecular mechanism underlying this modulation has not been fully resolved. On the contrary, initial reports of identification of key components in this modulation were later refuted using advanced model systems, especially transgenic animals. Some of the cardinal debated issues include details of specific subunits and residues in CaV1.2 phosphorylated by PKA, the nature, extent, and role of post-translational processing of CaV1.2, and the role of auxiliary proteins (such as A kinase anchoring proteins) involved in PKA regulation. In addition, the previously proposed crucial role of PKA in modulation of unstimulated Ca²âº current in the absence of ß-adrenergic receptor stimulation and in voltage-dependent facilitation of CaV1.2 remains uncertain. Full reconstitution of the ß-adrenergic receptor signaling pathway in heterologous expression systems remains an unmet challenge. This review summarizes the past and new findings, the mechanisms proposed and later proven, rejected or disputed, and emphasizes the essential issues that remain unresolved.

Competitive and non-competitive regulation of calcium-dependent inactivation in CaV1.2 L-type Ca2+ channels by calmodulin and Ca2+-binding protein 1.

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.

Early infantile epileptic encephalopathy associated with a high voltage gated calcium channelopathy.

BACKGROUND: Early infantile epileptic encephalopathies usually manifest as severely impaired cognitive and motor development and often result in a devastating permanent global developmental delay and intellectual disability. A large set of genes has been implicated in the aetiology of this heterogeneous group of disorders. Among these, the ion channelopathies play a prominent role. In this study, we investigated the genetic cause of infantile epilepsy in three affected siblings. METHODS AND RESULTS: Homozygosity mapping in DNA samples followed by exome analysis in one of the patients resulted in the identification of a homozygous mutation, p.L1040P, in the CACNA2D2 gene. This gene encodes the auxiliary α(2)δ2 subunit of high voltage gated calcium channels. The expression of the α(2)δ2-L1040P mutant instead of α(2)δ2 wild-type (WT) in Xenopus laevis oocytes was associated with a notable reduction of current density of both N (Ca(V)2.2) and L (Ca(V)1.2) type calcium channels. Western blot and confocal imaging analyses showed that the α(2)δ2-L1040P mutant was synthesised normally in oocyte but only the α(2)δ2-WT, and not the α(2)δ2-L1040P mutant, increased the expression of α(1B), the pore forming subunit of Ca(V)2.2, at the plasma membrane. The expression of α(2)δ2-WT with Ca(V)2.2 increased the surface expression of α(1B) 2.5-3 fold and accelerated current inactivation, whereas α(2)δ2-L1040P did not produce any of these effects. CONCLUSIONS: L1040P mutation in the CACNA2D2 gene is associated with dysfunction of α(2)δ2, resulting in reduced current density and slow inactivation in neuronal calcium channels. The prolonged calcium entry during depolarisation and changes in surface density of calcium channels caused by deficient α(2)δ2 could underlie the epileptic phenotype. This is the first report of an encephalopathy caused by mutation in the auxiliary α(2)δ subunit of high voltage gated calcium channels in humans, illustrating the importance of this subunit in normal physiology of the human brain.

Revealing the MRI-Contrast in Optically Cleared Brains.

The current consensus holds that optically-cleared specimens are unsuitable for Magnetic Resonance Imaging (MRI); exhibiting absence of contrast. Prior studies combined MRI with tissue-clearing techniques relying on the latter's ability to eliminate lipids, thereby fostering the assumption that lipids constitute the primary source of ex vivo MRI-contrast. Nevertheless, these findings contradict an extensive body of literature that underscores the contribution of other features to contrast. Furthermore, it remains unknown whether non-delipidating clearing methods can produce MRI-compatible specimens or whether MRI-contrast can be re-established. These limitations hinder the development of multimodal MRI-light-microscopy (LM) imaging approaches. This study assesses the relation between MRI-contrast, and delipidation in optically-cleared whole brains following different tissue-clearing approaches. It is demonstrated that uDISCO and ECi-brains are MRI-compatible upon tissue rehydration, despite both methods' substantial delipidating-nature. It is also demonstrated that, whereas Scale-clearing preserves most lipids, Scale-cleared brain lack MRI-contrast. Furthermore, MRI-contrast is restored to lipid-free CLARITY-brains without introducing lipids. Our results thereby dissociate between the essentiality of lipids to MRI-contrast. A tight association is found between tissue expansion, hyperhydration and loss of MRI-contrast. These findings then enabled us to develop a multimodal MRI-LM-imaging approach, opening new avenues to bridge between the micro- and mesoscale for biomedical research and clinical applications.

Datasets assessing lipid-content in optically cleared brains.

Multi-modal imaging, by light-microscopy (LM) and Magnetic Resonance Imaging (MRI), holds promise for examining the brain across various resolutions and scales. While MRI acquires images in three dimensions, acquisition of intact whole-brain by LM requires a process of tissue clearing that renders the brain transparent. Removal of lipids (delipidation) is a critical step in the tissue clearing process, and was previsouly suggested to be the cause for absence of MRI contrast in cleared brains. Yet, the association between MRI contrast, delipidation and the different clearing techniques is debatable. Here, we provide datasets concerning lipid-content in cleared brain tissues obtained by various approaches. Fixed mouse and rat brains were cleared by CLARITY, Scale, uDISCO and ECi clearing techniques. Lipid-content was assessed at various intermediate steps of the different clearing methods, as well as at the end of the processes. Methods employed included whole brain MRI acquisition, Oil Red O (ORO)- and carbocyanine DiI-staining of cryosections, and DiI-washout assay from brain slices. MRI contrast-to-noise ratio, staining intensities and integrity of tissue were systematically analyzed. We demonstrate that lipid electrophoresis, an essential step of the CLARITY approach, engenders progressive reduction in MRI contrast in non-cleared (PFA-fixed) control brains, as well as strongly reduces contrast from uDISCO and ECi-cleared brains. ORO minimally stained CLARITY-cleared brains, however efficiently labelled uDISCO and ECi-cleared brains. Conversely, and in contrast to ORO-staining, DiI equally stained control, CLARITY, ECi and uDISCO-cleared brains. Both ORO- and DiI-staining demonstrated impairment in brain tissue integrity following CLARITY, but less so in uDISCO and ECi brains. DiI-washout assay demonstrated that each of the solvents employed along the process of uDISCO and ECi are highly delipidating, as well as the SDS-electrophoresis employed during CLARITY clearing. However, Scale treatment preserved most of the DiI dye. These data emphasize the variability in lipid assessment of cleared tissues by common techniques, and may help to resolve the contribution of lipids in brain MRI contrast.

CaBP1 regulates voltage-dependent inactivation and activation of Ca(V)1.2 (L-type) calcium channels.

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).

Andersen-Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1.

Andersen-Tawil syndrome (ATS) type-1 is associated with loss-of-function mutations in KCNJ2 gene. KCNJ2 encodes the tetrameric inward-rectifier potassium channel Kir2.1, important to the resting phase of the cardiac action potential. Kir-channels' activity requires interaction with the agonist phosphatidylinositol-4,5-bisphosphate (PIP2). Two mutations were identified in ATS patients, V77E in the cytosolic N-terminal "slide helix" and M307V in the C-terminal cytoplasmic gate structure "G-loop." Current recordings in Kir2.1-expressing HEK cells showed that each of the two mutations caused Kir2.1 loss-of-function. Biotinylation and immunostaining showed that protein expression and trafficking of Kir2.1 to the plasma membrane were not affected by the mutations. To test the functional effect of the mutants in a heterozygote set, Kir2.1 dimers were prepared. Each dimer was composed of two Kir2.1 subunits joined with a flexible linker (i.e. WT-WT, WT dimer; WT-V77E and WT-M307V, mutant dimer). A tetrameric assembly of Kir2.1 is expected to include two dimers. The protein expression and the current density of WT dimer were equally reduced to ~25% of the WT monomer. Measurements from HEK cells and Xenopus oocytes showed that the expression of either WT-V77E or WT-M307V yielded currents of only about 20% compared to the WT dimer, supporting a dominant-negative effect of the mutants. Kir2.1 sensitivity to PIP2 was examined by activating the PIP2 specific voltage-sensitive phosphatase (VSP) that induced PIP2 depletion during current recordings, in HEK cells and Xenopus oocytes. PIP2 depletion induced a stronger and faster decay in Kir2.1 mutant dimers current compared to the WT dimer. BGP-15, a drug that has been demonstrated to have an anti-arrhythmic effect in mice, stabilized the Kir2.1 current amplitude following VSP-induced PIP2 depletion in cells expressing WT or mutant dimers. This study underlines the implication of mutations in cytoplasmic regions of Kir2.1. A newly developed calibrated VSP activation protocol enabled a quantitative assessment of changes in PIP2 regulation caused by the mutations. The results suggest an impaired function and a dominant-negative effect of the Kir2.1 variants that involve an impaired regulation by PIP2. This study also demonstrates that BGP-15 may be beneficial in restoring impaired Kir2.1 function and possibly in treating ATS symptoms.

Reduction in Filamin C transcript is associated with arrhythmogenic cardiomyopathy in Ashkenazi Jews.

BACKGROUND: Filamin C is a cytoskeletal protein expressed in cardiac cells. Nonsense variations in the filamin C gene (FLNC) were associated with dilated and arrhythmogenic cardiomyopathies. METHODS AND RESULTS: We identified an intronic variation in FLNC gene (c.3791-1G > C) in three unrelated Ashkenazi Jewish families with variable expression of arrhythmia and cardiomyopathy. cDNA was prepared from a mutation carrier's cultured skin fibroblasts. Quantitative PCR demonstrated a reduction in total FLNC transcript, and no other FLNC splice variants were found. Single-nucleotide polymorphism (SNP) analysis revealed heterozygous variations in the genomic DNA that were not expressed in the messenger RNA. Immunohistochemical analysis of cardiac sections detected a normal distribution of filamin C protein in the heart ventricles. CONCLUSION: The transcript that included the FLNC variant was degraded. Haploinsufficiency in filamin C underlies arrhythmogenic cardiomyopathy with variable symptoms.

Mutations in NaV1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel.

Mutations in the SCN5A gene, encoding the cardiac voltage-gated sodium channel NaV1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca2+-dependent mechanisms and the involvement of ß-subunit (NaVß) in NaV1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the SCN5A gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of NaV1.5, both are putative CaM-binding domains. These mutants were functionally studied in human embryonic kidney (HEK) cells and HL-1 cells using whole-cell patch clamp technique. Calmodulin (CaM) interaction and cell-surface expression of heterologously expressed NaV1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered NaV1.5 non-conductive. NaV1.5K1493del altered the gating properties of co-expressed functional NaV1.5, in a Ca2+ and NaVß1-dependent manner. NaV1.5A1924T impaired NaVß1-dependent gating regulation. Ca2+-dependent CaM-interaction with NaV1.5 was blunted in NaV1.5K1493del. Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca2+-dependent gating regulation of NaV1.5 channels. Our results highlight the role of NaV1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca2+-sensing machinery of NaV1.5 involves NaVß1.

Modulation of distinct isoforms of L-type calcium channels by G(q)-coupled receptors in Xenopus oocytes: antagonistic effects of Gßγ and protein kinase C.

L-type voltage dependent Ca(2+) channels (L-VDCCs; Ca(v)1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G(q) enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G(q)-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α(1C), whereas known smooth muscle short-NT isoforms were inhibited by PKC and G(q) activators. We report a novel regulation of the long-NT α(1C) isoform by Gßγ. Gßγ inhibited whereas a Gßγ scavenger protein augmented the G(q)--but not phorbol ester-mediated enhancement of channel activity, suggesting that Gßγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gßγ and PKC to α(1C)-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α(1C) sensitive to PKC stimulation and to Gßγ scavenging. Our results suggest a complex antagonistic interplay between G(q)-activated PKC and Gßγ in regulation of L-VDCC, in which multiple cytosolic segments of α(1C) are involved.

Characterization of the calmodulin-binding site in the N terminus of CaV1.2.

Interaction of calmodulin (CaM) with the C-terminus (CT) of the L-type Ca(V)1.2 channel is crucial for Ca(2+)-dependent inactivation (CDI). CaM also binds to the N-terminus (NT), and a CaM-formed "bridge" between CT and NT has been proposed to control CDI. We characterized the interaction of CaM with its NT-binding peptide. Binding is Ca(2+)-dependent with an affinity of 0.6 microM. Mutations in NT of Ca(V)1.2 that abolished the binding of CaM only slightly weakened the CDI but also accelerated the VDI. CaM did not foster an interaction between the CaM-binding peptides of NT and CT. Thus, the role of CaM's interaction with the Ca(V)1.2 NT remains to be determined.