Mutations In Ca2 Research Articles

High-yield Heterologous Expression of Wild Type and Mutant Ca 2+ ATPase: Characterization of Ca 2+ Binding Sites by Charge Transfer

High-yield heterologous SERCA1 (Ca 2+ ATPase) expression was obtained in COS-1 cells infected with recombinant adenovirus vector (rAdSERCA). Higher transcription and expression were obtained in the presence of a His 6 tag at the amino terminus, as compared with a His 6 tag at the carboxyl SERCA terminus, or no tag. The expressed protein was targeted extensively to intracellular membranes. Optimal yield of functional Ca 2+ ATPase corresponded to 10% of total protein, with phosphoenzyme levels, catalytic turnover and Ca 2+ transport identical with those of native SERCA1. This recombinant membrane-bound (detergent-free) enzyme was used for characterization of Ca 2+ binding at the two specific transmembrane sites (ATP-free) by measurements of net charge transfer upon Ca 2+ binding to the protein, yielding cooperative isotherms ( K 1 = 5.9 ± 0.5 × 10 5 M −1 and K 2 = 5.7 ± 0.3 × 10 6 M −1). Non-cooperative binding of only one Ca 2+, and loss of ATPase activation, were observed following E309 mutation at site II. On the other hand, as a consequence of the site II mutation, the affinity of site I for Ca 2+ was increased ( K = 4.4 ± 0.2 × 10 6 M −1). This change was due to a p K a shift of site I acidic residues, and to contributions of oxygen functions from empty site II to Ca 2+ binding at site I. No charge movement was observed following E771Q mutation at site I, indicating no Ca 2+ binding to either site. Therefore, calcium occupancy of site I is required to trigger cooperative binding to site II and catalytic activation. In the presence of millimolar Mg 2+, the charge movement upon addition of Ca 2+ to WT ATPase was reduced by 50%, while it was reduced by 90% when Ca 2+ was added to the E309Q/A mutants, demonstrating that competitive Mg 2+ binding can occur at site I but not at site II.

Characterization of Calmodulin with Mutated Ca2+-Binding Sites

Calmodulin (CaM) regulates cellular functions via its Ca2+ binding properties. The N- and C-domains of CaM, which are separated by a flexible tether, each bind two Ca2+ ions via EF-hand motifs. Mutation of position 1 in individual EF hands (the X coordination site) from Asp to Ala has been used to selectively inhibit Ca2+ binding to the N- and C-domains of CaM. We used this mutation strategy to investigate how the individual Ca2+ binding sites contribute to the association of PEP-19 with CaM. Four CaM mutants were made and designated CaM12, CaM3, CaM4 and CaM34 based on nomenclature established in the literature. Ideally, all mutant proteins should be structurally and functionally identical to native CaM in the absence of Ca2+, however, mutation of Ca2+ binding sites in the C-domain of CaM caused weak affinity and significantly different koff and kon rates for binding PEP-19. This led us to use NMR and other methods to determine the degree of structural perturbation caused by mutation of the EF-hands. Inhibition of Ca2+ binding to the N-domain in CaM12 does not affect Ca2+ binding to the C-domain, however, inhibition of Ca2+-binding to the C-domain in CaM3 and CaM34 significantly increases the Ca2+-binding affinity of the N-domain by decreasing the koff for Ca2+. This was associated with increased exposure of hydrophobic regions in the N-domain as detected by ANS fluorescence. Significantly, 1H-15N HSQC spectra collected in the absence of Ca2+ show large structural perturbations in the C-domain of CaM3, CaM4 and especially CaM34 relative to apo-CaM. This was observed as resonance broadening and a loss of dispersion. These data indicate that conversion of Asp 93 and 129 to Ala destabilizes the C-domain of apo-CaM.

Alteration of Sarcoplasmic ReticulumCa2+Release in Skeletal Muscle from Calpain 3-Deficient Mice

Mutations of Ca2+-activated proteases (calpains) cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR) agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC) suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR) Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

Expression and 1,4-Dihydropyridine-Binding Properties of Brain L-Type Calcium Channel Isoforms

The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.

Toxicity and Endocytosis of Spinocerebellar Ataxia Type 6 Polyglutamine Domains: Role of Myosin IIB†

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease caused by a small expansion of CAG repeats in the sequence coding for the cytoplasmic C-terminal region of the Ca(v)2.1 subunit of P/Q-type calcium channels. We have tested the toxicity of mutated Ca(v)2.1 C-terminal domains expressed in the plasma membrane. In COS-7 cells, CD4-green fluorescent protein fused to Ca(v)2.1 C-terminal domains containing expanded 24 polyglutamine (Q) tracts displayed increased toxicity and stronger expression at the cell surface relative to 'normal' 12 Q tracts, partially because of reduced endocytosis. Glutathione S-transferase pull-down and proteomic analysis indicated that Ca(v)2.1 C-termini interact with the heavy and light chains of cerebellar myosin IIB, a molecular motor protein. This interaction was confirmed by coimmunoprecipitation from rat cerebellum and COS-7 cells and shown to be direct by binding of in vitro-translated (35)S-myosin IIB heavy chain. In COS-7 cells, incremented polyglutamine tract length increased the interaction with myosin IIB. Furthermore, the myosin II inhibitor blebbistatin reversed the effects of polyglutamine expansion on plasma membrane expression. Our findings suggest a key role of myosin IIB in promoting accumulation of mutant Ca(v)2.1Ct at the plasma membrane and suggest that this gain of function might contribute to the pathogenesis of SCA6.

The S218L familial hemiplegic migraine mutation promotes deinhibition of Cav2.1 calcium channels during direct G-protein regulation

Familial hemiplegic migraine type 1 (FHM-1) is caused by mutations in CACNA1A, the gene encoding for the Ca(v)2.1 subunit of voltage-gated calcium channels. Although various studies attempted to determine biophysical consequences of these mutations on channel activity, it remains unclear exactly how mutations can produce a FHM-1 phenotype. A lower activation threshold of mutated channels resulting in increased channel activity has been proposed. However, hyperactivity may also be caused by a reduction of the inhibitory pathway carried by G-protein-coupled-receptor activation. The aim of this study is to determine functional consequences of the FHM-1 S218L mutation on direct G-protein regulation of Ca(v)2.1 channels. In HEK 293 cells, DAMGO activation of human mu-opioid receptors induced a 55% Ba(2+) current inhibition through both wild-type and S218L mutant Ca(v)2.1 channels. In contrast, this mutation considerably accelerates the kinetic of current deinhibition following channel activation by 1.7- to 2.3-fold depending on membrane potential values. Taken together, these data suggest that the S218L mutation does not affect G-protein association onto the channel in the closed state but promotes its dissociation from the activated channel, thereby decreasing the inhibitory G-protein pathway. Similar results were obtained with the R192Q FHM-1 mutation, although of lesser amplitude, which seems in line with the less severe associated clinical phenotype in patients. Functional consequences of FHM-1 mutations appear thus as the consequence of the alteration of both intrinsic biophysical properties and of the main inhibitory G-protein pathway of Ca(v)2.1 channels. The present study furthers molecular insight in the physiopathology of FHM-1.

Reduced ACh release at neuromuscular synapses of heterozygous leaner Cav2.1‐mutant mice

Episodic ataxia type 2 (EA2) is an autosomal dominantly inherited neurological disorder. Patients have CACNA1A gene mutations resulting in truncation or single amino acid changes in the pore-forming subunit of Ca(v)2.1 (P/Q-type) Ca(2+) channels. These neuronal channels mediate synaptic neurotransmitter release. EA2 symptoms are thought to result from disturbed neurotransmission at cerebellar and neuromuscular synapses, caused by loss-of-function of Ca(v)2.1 channels. Heterozygous leaner (Ln/wt) mice, carrying a Cacna1a truncation mutation, as well as heterozygous Ca(v)2.1 null-mutant (KO/wt) mice may model synaptic aspects of EA2. We studied Ca(v)2.1-mediated acetylcholine (ACh) release at their neuromuscular junctions (NMJs) ex vivo. KO/wt mice did not show any ACh release abnormalities, not even at older age. However, Ln/wt mice had approximately 25% reduced spontaneous uniquantal ACh release and approximately 10% reduced nerve-stimulation evoked release, compared with wild-type. EA2 is treated with acetazolamide (AZA), but the pharmacotherapeutic mechanism is unknown. We tested the possibility of a direct influence on (mutant) presynaptic Ca(v)2.1 channel function by studying the acute effect of 50 muM AZA on ACh release at ex vivo NMJs of wild-type, KO/wt, and Ln/wt mice. No changes were found in any of the release parameters. Our results indicate that Ln-mutated Ca(v)2.1 channels at Ln/wt NMJs are either normally inserted in the presynaptic membrane but have reduced function, or that they inhibit wild-type channels by hampering their expression, trafficking, membrane insertion and/or function. In this respect Ln/wt NMJs may model EA2 synapses. Furthermore, AZA does not exert an acute, direct influence on the function of presynaptic (mutant) Ca(v)2.1 channels.

Adenoviral-mediated expression of dihydropyridine-insensitive L-type calcium channels in cardiac ventricular myocytes and fibroblasts

Cardiac voltage-gated Ca 2+ channels regulate the intracellular Ca 2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca 2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive Ca V1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant Ca V1.2 channel (Ca V1.2DHP i) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca 2+ current ( I Ca) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC 50 of 66 nM and complete block at 250 nM. In contrast, I Ca in cells infected with AdCa V1.2DHP i was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 μM diltiazem. In order to study Ca V1.2DHP i in isolation, myocytes infected with AdCa V1.2DHP i were incubated with nisoldipine. Under this condition the cells expressed a large I Ca (12 pA/pF) and displayed Ca 2+ transients during field stimulation. Furthermore, addition of 2 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased I Ba in the AdCa V1.2DHP i-infected cells. A Cd 2+-sensitive I Ba was also recorded in cardiac fibroblasts infected with AdCa V1.2DHP i. Thus, expression of Ca V1.2DHP i will provide an important tool in studies of cardiac myocyte and fibroblast function.

Pain sensitivity in mice lacking the Ca v2.1α 1 subunit of P/Q-type Ca 2+ channels

The role of voltage-gated Ca 2+ (Ca V) channels in pain mechanisms has been the object of intense investigation using pharmacological approaches and, more recently, using mutant mouse models lacking the Ca Vα l pore-forming subunit of N-, R- and T-type channels. The role of P/Q-type channels in nociception and pain transmission has been investigated by pharmacological approaches but remains to be fully elucidated. To address this issue, we have analyzed pain-related behavioral responses of null mutant mice for the Ca V2.1α 1 subunit of P/Q-type channels. Homozygous null mutant Ca V2.1α 1−/− mice developed dystonia at 10–12 days after birth and did not survive past weaning. Tested at ages where motor deficit was either absent or very mild, Ca V2.1α 1−/− mice showed reduced tail withdrawal latencies in the tail-flick test and reduced abdominal writhes in the acetic acid writhing test. Adult heterozygous Ca V2.1α 1+/− mice did not show motor deficits in the rotarod and activity cage tests and did not show alterations in pain responses in the tail-flick test and the acetic acid writhing test. Strikingly, they showed a reduced licking response during the second phase of formalin-induced inflammatory pain and a reduced mechanical allodynia in the chronic constriction injury model of neuropathic pain. Our findings show that P/Q-type channels play an antinociceptive role in sensitivity to non-injurious noxious thermal stimuli and a pronociceptive role in inflammatory and neuropathic pain states, pointing to an important role of Ca V2.1 channels in central sensitization.

Phosphorylation sites on calcium channel α1 and β subunits regulate ERK-dependent modulation of neuronal N-type calcium channels

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca v2.2) calcium channels and all four neuronal calcium channel β subunits (Ca vβ), suggests that Ca v2.2 and/or Ca vβs may be ERK-phosphorylated. Here we report that GST-Ca v2.2 I–II loop, and to a lesser extent Ca vβ1b-His 6, are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca v2.2 I–II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca v2.2,β1b,α2δ1 versus mutant Ca v2.2(S447A) or Ca v2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca vβ1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca v2.2(S447A) with Ca vβ1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca vβ1b(S161,348A) was co-expressed with Ca v2.2(S409A). Thus, Ser-447 on Ca v2.2 and Ser-161 and Ser-348 of Ca vβ1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca v2.2α1 and to a lesser extent possibly also Ca vβ subunits.

Genetically Encoded Bright Ca2+ Probe Applicable for Dynamic Ca2+ Imaging of Dendritic Spines

G-CaMP is a Ca2+ probe based on a single green fluorescent protein (GFP). G-CaMP shows a large fluorescence increase upon Ca2+ binding, but its fluorescence is dim and pH sensitive, similar to other single GFP-based probes. Here we report an improved G-CaMP, named G-CaMP1.6, which enables easier detection of intracellular Ca2+ signals. G-CaMP1.6 was approximately 40 times more fluorescent than G-CaMP, mainly due to an increase in quantum yield. Furthermore, compared with G-CaMP, G-CaMP1.6 had not only a lower pH sensitivity but also a higher selectivity for divalent cations having an ionic radius similar to Ca2+. Ca2+ sensitivity of G-CaMP1.6 (Kd = 146 nM, Hill coefficient = 3.8, Fmax/Fmin = 4.9) was slightly shifted toward higher affinity compared with that of G-CaMP. When expressed in mammalian cells, G-CaMP1.6 showed large fluorescence changes with drug applications. Notably, local Ca2+ changes in such tiny structures as dendritic spines of neurons were successfully observed with G-CaMP1.6, this being the first observation using a GFP-based probe. Additional mutations in Ca2+-binding sites of G-CaMP1.6 shifted the affinity for Ca2+ and reduced the Ca2+-buffering effect. G-CaMP1.6-CaM(E140K), which has a mutation in the Ca2+ binding site, is an improved probe with its increased brightness and reduced Ca2+-buffering capacity.

Reverse engineering the L-type Ca 2+ channel α 1c subunit in adult cardiac myocytes using novel adenoviral vectors

The α 1c subunit of the cardiac L-type Ca 2+ channel, which contains the channel pore, voltage- and Ca 2+-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca 2+ channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length α 1c gene cassettes into the adenovirus backbone. Wild-type (α 1c-wt) and mutant (α 1c-D-) Ca 2+ channel adenoviruses were constructed. The α 1c-D- contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α 1c-wt and α 1c-D- expressed robustly in A549 cells (peak L-type Ca 2+ current ( I CaL) at 0 mV: α 1c-wt −9.94 ± 1.00 pA/pF, n = 9; α 1c-D- −10.30 pA/pF, n = 12). I CaL carried by α 1c-D- was markedly less sensitive to nitrendipine (IC 50 17.1 μM) than α 1c-wt (IC 50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 μM nitrendipine blocked only 51 ± 5% ( n = 9) of I CaL in α 1c-D--expressing myocytes, in comparison to 86 ± 8% ( n = 9) of I CaL in control myocytes. Moreover, in 20 μM nitrendipine, calcium transients could still be evoked in α 1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca 2+ release. These α 1c adenoviruses provide an unprecedented tool for structure–function studies of cardiac excitation–contraction coupling and L-type Ca 2+ channel regulation in the native myocyte background.

Smooth muscle cell expression of a constitutive active form of human Rac 1 accelerates cutaneous wound repair

Hyperoxia has been shown to improve wound healing; however, the mechanism for such therapeutic effects of oxygen remains hypothetical. Rac 1 regulates a wide variety of cellular activities, including cell proliferation and migration, and also is a key regulator for the activity of the nicotinamide dinucleotide phosphate oxidase the enzyme complex responsible for the production of a large fraction of cellular superoxide. We generated transgenic mice that express either the cDNA of a constitutively active mutant of human Rac 1 (V12 mutant or Rac CA) or the dominant negative isoform (V12 and N17 mutant or Rac DN) in the blood vessels using mouse vascular smooth muscle promoter for alpha-actin. We placed 2 wounds of 6 mm in diameter at the middorsal region of each mouse and allowed about 3 weeks for the wounds to heal. The size of the wounds in Rac CA transgenic mice was reduced relative to wild type mice; healing of Rac DN mice was slower than wild type and Rac CA ( P < .05). Blood vessel formation appeared faster in Rac CA mice, a finding associated with enhanced expression of some angiogenic growth factors. The current studies suggest that Rac 1 activation accelerates the wound healing process and is associated with more efficient angiogenesis at the wound site.

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2.

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.

Carbonic anhydrase II deficiency syndrome (osteopetrosis with renal tubular acidosis and brain calcification): novel mutations in CA2 identified by direct sequencing expand the opportunity for genotype-phenotype correlation.

The carbonic anhydrase II (CA II) deficiency syndrome is an autosomal recessive disorder that produces osteopetrosis, renal tubular acidosis, and cerebral calcification. Other features include developmental delay, short stature, cognitive defects, and a history of multiple fractures by adolescence. With one exception, all patients with osteopetrosis and renal tubular acidosis examined have proven to have CA II deficiency. All CA II-deficient patients analyzed have been found to have mutations in the CA2 gene. Previously, we used single strand conformational (SSCP) analysis to identify exons to be sequenced from CA II-deficient patients. In this report, we amplified all seven exons by PCR from genomic DNA and directly sequenced the amplified products. Application of this method allowed identification of eleven new mutations in 21 patients referred for confirmation of the diagnosis of CA II deficiency. These mutations were scattered over the genome from exon 2 to 7. In two opportunities for prenatal diagnosis, one from cultured amniocytes and one from chorionic villus biopsy, we demonstrated the general utility of the direct sequencing method for prenatal DNA diagnosis. These studies expand our knowledge of the heterogeneity in mutations underlying the CA II deficiency syndrome.

Analysis of Oxygen-Sensitive Human Cardiac L-Type Ca2+ Channel α1C Subunit (hHT Isoform)

Publisher Summary This chapter discusses the analysis of oxygen-sensitive human cardiac L-type Ca 2+ channel α 1C subunit (hHT isoform). A study is described, which demonstrated the hypoxic inhibition of a recombinant human cardiac L-type Ca 2+ channel α1C subunit (the hHT splice variant). Inhibition was observed following stable α1C expression in HEK293 cells, in the absence of auxiliary subunits, and the effects of hypoxia appeared to mimic perfectly the effects of hypoxia on native L-type Ca 2+ channel activity in smooth muscle cells. In this chapter, the techniques used to generate mutant L-type Ca 2+ channels and to record their responses to hypoxia following expression in HEK293 cells are discussed. Experimental methodology for the construction of mutant L-type Ca 2+ channels, swapping C-tail segments between hHT and rHT cDNA clones, experimental procedures for blue–white colony screening (colorimetric assay) and purification and concentration of DNA are also described.

Molecular and pharmacological bases for the gating regulation of L-type voltage-dependent Ca2+ channels

The voltage-dependent L-type Ca(2+) channel plays a key role in the spacial and temporal regulation of Ca(2+). In cardiac excitation-contraction coupling, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), triggered by Ca(2+) entry through the nearby L-type Ca(2+) channel, induces the Ca(2+)-dependent inactivation (CDI) of the Ca(2+) channel. We demonstrated that the CICR-dependent CDI of L-type Ca(2+) channels, under control of the privileged cross-signaling between L-type Ca(2+) channels and RyRs, plays important roles for monitoring and tuning the SR Ca(2+) content via changes of AP waveform and the amount of Ca(2+)-influx during AP in ventricular myocytes. L-type Ca(2+) channels are modulated by the binding of Ca(2+) channel antagonists and agonists to the pore-forming alpha(1C) subunit. We identified Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region of the alpha(1C) subunit as critical determinants of the binding of dihydropyridines (DHP). Interestingly, double mutant Ca(2+) channel (F1112A/S1115A) failed to discriminate between a DHP Ca(2+) channel agonist and antagonist stereoisomers. We proposed that Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region is required for the stabilization of the Ca(2+) channel in the open state by Ca(2+) channel agonists and further proposed a novel model for the DHP-binding pocket of the alpha(1C) subunit. These integrative studies on the gating regulation of cardiac L-type Ca(2+) channels will provide the molecular basis for the pharmacology of Ca(2+) channel modulators.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion.

Exocytosis-the release of the contents of a vesicle--proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Functional Roles of Ca v 1.3 (α 1D ) Calcium Channel in Sinoatrial Nodes

We directly examined the role of the Ca v 1.3 (α 1D ) Ca 2+ channel in the sinoatrial (SA) node by using Ca v 1.3 Ca 2+ channel-deficient mice. A previous report has shown that the null mutant (Ca v 1.3 −/− ) mice have sinus bradycardia with a prolonged PR interval. In the present study, we show that spontaneous action potentials recorded from the SA nodes show a significant decrease in the beating frequency and rate of diastolic depolarization in Ca v 1.3 −/− mice compared with their heterozygous (Ca v 1.3 +/− ) or wild-type (WT, Ca v 1.3 +/+ ) littermates, suggesting that the deficit is intrinsic to the SA node. Whole-cell L-type Ca 2+ currents ( I Ca,L s) recorded in single isolated SA node cells from Ca v 1.3 −/− mice show a significant depolarization shift in the activation threshold. The voltage-dependent activation of Ca v 1.2 (α 1C ) versus Ca v 1.3 Ca 2+ channel subunits was directly compared by using a heterologous expression system without β coexpression. Similar to the I Ca,L recorded in the SA node of Ca v 1.3 −/− mutant mice, the Ca v 1.2 Ca 2+ channel shows a depolarization shift in the voltage-dependent activation compared with that in the Ca v 1.3 Ca 2+ channel. In summary, using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we were able to establish a role for Ca v 1.3 Ca 2+ channels in the generation of the spontaneous action potential in SA node cells.

Involvement of the Cytoplasmic Loop L6–7 in the Entry Mechanism for Transport of Ca2+ through the Sarcoplasmic Reticulum Ca2+-ATPase

We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.