Mutations in IMPG1 Cause Vitelliform Macular Dystrophies

Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507^∗). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD.     

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (nu) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be approximately 138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.     

Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels

Genetic analyses have revealed an association between the gene encoding the Rab3A-interacting molecule (RIM1) and the autosomal dominant cone-rod dystrophy CORD7. However, the pathogenesis of CORD7 remains unclear. We recently revealed that RIM1 regulates voltage-dependent Ca(2+) channel (VDCC) currents and anchors neurotransmitter-containing vesicles to VDCCs, thereby controlling neurotransmitter release. We demonstrate here that the mouse RIM1 arginine-to-histidine substitution (R655H), which corresponds to the human CORD7 mutation, modifies RIM1 function in regulating VDCC currents elicited by the P/Q-type Ca(v)2.1 and L-type Ca(v)1.4 channels. Thus, our data can raise an interesting possibility that CORD7 phenotypes including retinal deficits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents.     

Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel

The importance of voltage-dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N-type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain-related phenomena. To understand the genuine contribution of the N-type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N-type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N-type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.     

Distinct post-translational modifications regulate BK channel activity: The interplay between protein palmitoylation and phosphorylation

Genetic analyses have revealed an association between the gene encoding the Rab3A?interacting molecule (RIM1) and the autosomal dominant cone?rod dystrophy CORD7. However, the pathogenesis of CORD7 remains unclear. We recently revealed that RIM1 regulates voltage?dependent Ca2+ channel (VDCC) currents and anchors neurotransmitter?containing vesicles to VDCCs, thereby controlling neurotransmitter release. We demonstrate here that the mouse RIM1 arginine?to?histidine substitution (R655H), which corresponds to the human CORD7 mutation, modifies RIM1 function in regulating VDCC currents elicited by the P/Q?type Cav2.1 and L?type Cav1.4 channels. Thus, our data can raise an interesting possibility that CORD7 phenotypes including retinal deficits and enhanced cognition are at least partly due to altered regulation of presynaptic VDCC currents.

Addendum to:

RIM1 Confers Sustained Activity and Neurotransmitter Vesicle Anchoring to Presynaptic Ca2+ Channels

S. Kiyonaka, M. Wakamori, T. Miki, Y. Uriu, M. Nonaka, H. Bito, A.M. Beedle, E. Mori, Y. Hara, M. De Waard, M. Kanagawa, M. Itakura, M. Takahashi, K.P. Campbell, and Y. Mori

Nat Neurosci 2007; 10:691-701     

Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel

In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca(2+)-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca(2+) channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca(2+) influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC β(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC β(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC α(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of β(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.     

The lipid peroxidation product 4-hydroxynonenal facilitates opening of voltage-dependent Ca2+ channels in neurons by increasing protein tyrosine phosphorylation

Calcium influx through voltage-dependent calcium channels (VDCCs) mediates a variety of functions in neurons and other excitable cells, but excessive calcium influx through these channels can contribute to neuronal death in pathological settings. Oxyradical production and membrane lipid peroxidation occur in neurons in response to normal activity in neuronal circuits, whereas excessive lipid peroxidation is implicated in the pathogenesis of of neurodegenerative disorders. We now report on a specific mechanism whereby lipid peroxidation can modulate the activity of VDCCs. The lipid peroxidation product 4-hydroxy-2,3-nonenal (4HN) enhances dihydropyridine-sensitive whole-cell Ca2+ currents and increases depolarization-induced increases of intracellular Ca2+ levels in hippocampal neurons. Prolonged exposure to 4HN results in neuronal death, which is prevented by treatment with glutathione and attenuated by the L-type Ca2+ channel blocker nimodipine. Tyrosine phosphorylation of alpha1 VDCC subunits is increased in neurons exposed to 4HN, and studies using inhibitors of tyrosine kinases and phosphatases indicate a requirement for tyrosine phosphorylation in the enhancement of VDCC activity in response to 4HN. Phosphorylation-mediated modulation of Ca2+ channel activity in response to lipid peroxidation may play important roles in the responses of neurons to oxidative stress in both physiological and pathological settings.     

Essential role of ubiquitin-proteasome system in normal regulation of insulin secretion

Insulin secretion from pancreatic beta-cells occurs by sequential cellular processes, including glucose metabolism, electrical activity, Ca2+ entry, and regulated exocytosis. Abnormalities in any of these functions can impair insulin secretion. In the present study, we demonstrate that inhibition of proteasome activity severely reduces insulin secretion in the mouse pancreatic beta-cell line MIN6-m9. Although no significant effects on glucose metabolism including ATP production were found in the presence of proteasome inhibitors, both glucose- and KCl-induced Ca2+ entry were drastically reduced. As Ca2+-ionophore-induced insulin secretion was unaffected by proteasome inhibition, a defect in Ca2+ entry through voltage-dependent calcium channels (VDCCs) is the likely cause of the impaired insulin secretion. We found that the pore-forming alpha-subunit of VDCCs undergoes ubiquitination, which does not decrease but slightly increases expression of the alpha-subunit protein at the plasma membrane. However, electrophysiological analysis revealed that treatment with proteasome inhibitors results in a severe reduction in VDCC activity in MIN6-m9 cells, indicating that VDCC function is suppressed by proteasome inhibition. Furthermore, insulin secretion in isolated mouse pancreatic islets was also decreased by proteasome inhibition. These results demonstrate that the ubiquitin-proteasome system plays a critical role in insulin secretion by maintaining normal function of VDCCs.     

Functional analysis of a rice putative voltage-dependent Ca2+ channel, OsTPC1, expressed in yeast cells lacking its homologous gene CCH1

We isolated a cDNA (OsTPC1) from rice that was homologous to AtTPC1, a putative voltage-dependent Ca(2+) channel (VDCC) gene of Arabidopsis thaliana. The hydropathy profile of its deduced amino acid sequence showed significant structural features of the alpha 1-subunit of animal VDCCs. Functional analysis using a heterologous yeast expression system showed that OsTPC1 facilitates Ca(2+) permeation. The K(m) value for Ca(2+) of OsTPC1, 47.5 micro M, was comparable to that of intrinsic CCH1, a candidate VDCC in yeast. Ca(2+) permeation by OsTPC1 was inhibited by verapamil, a VDCC blocker. These findings indicate for the first time that OsTPC1 is a putative VDCC in rice.     

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl⁻ channel and/or a regulator of Ca⁺⁺ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S_20,w) of 4.9, and partial specific volume (ν) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be ∼138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (V_o) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.     

Direct inhibition of the interaction between alpha-interaction domain and beta-interaction domain of voltage-dependent Ca2+ channels by Gem

The Ras-related small G-protein Gem regulates voltage-dependent Ca2+ channels (VDCCs) through interaction with the beta-subunit of the VDCC. This action of Gem is mediated by regulated alpha1-subunit expression at the plasma membrane. In the present study, we examined the mechanism of the inhibition of VDCC activity by Gem. The beta-interaction domain (BID) of the beta-subunit, which specifically interacts with the alpha-interaction domain (AID) of the alpha1-subunit, is shown to be essential for the interaction between Gem and beta-subunits. In addition, the AID peptide inhibited interaction between Gem and beta-subunits in a dose-dependent manner. GemS88N mutant, which has low binding affinity for guanine nucleotide, did not interact with beta-subunits, allowing alpha1-subunit expression at the plasma membrane. This inhibitory effect of wild-type Gem on VDCC activity was reduced in cells expressing GemS88N. The overexpression of wild-type Gem in pancreatic beta-cell line MIN6 cells suppressed Ca2+-triggered secretion, whereas overexpression of GemS88N induced Ca2+-triggered secretion to control level. These results suggest that GTPase activity of Gem is required for the binding of Gem to BID that regulates VDCC activity through interaction with AID.     

Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation

Voltage-gated Ca2+ channels (VDCCs) are heteromultimeric proteins that mediate Ca2+ influx into cells upon membrane depolarization. These channels are involved in various cellular events, including gene expression, regulation of hormone secretion and synaptic transmission. Kir/Gem, Rad, Rem, and Rem2 belong to the RGK family of Ras-related small G proteins. RGK proteins interact with the beta-subunits and downregulate VDCC activity. Kir/Gem was proposed to prevent surface expression of functional Ca2+ channels, while for Rem2 the mechanism remains controversial. Here, we have analyzed the mechanism by which Rad and Rem regulate VDCC activity. We show that, similar to Kir/Gem and Rem2, 14-3-3 and CaM binding regulate the subcellular distribution of Rad and Rem, which both inhibit Ca2+ channel activity by preventing its expression on the cell surface. This function is regulated by calmodulin and 14-3-3 binding only for Rad and not for Rem. Interestingly, nuclear targeting of Rad and Rem can relocalize and sequester the beta-subunit to the nucleus, thus providing a novel mechanism for Ca2+ channel downregulation.     

Calcium channels involved in neurotransmitter release at adult,neonatal and P/Q-type deficient neuromuscular junctions (Review)

Different types of voltage-dependent calcium channels (VDCCs) have been recognized based on their molecular structure as well as their pharmacological and biophysical properties. One of these, the P/Q type, is the main channel involved in nerve evoked neurotransmitter release at neuromuscular junctions (NMJs) and many central nervous system synapses. However, under particular experimental or biological conditions, other channels can be involved. L-type VDCC presence at the NMJ has been demonstrated by the contribution to the perineural calcium currents (I Ca ) at adult mice Bapta-loaded NMJs. This is probably a result of a reduction in Ca 2+ inactivation. The L-type current was not coupled to neurotransmitter release, but became coupled, as demonstrated by the release of acetylcholine, after the inhibition of serine/threonine protein phosphatases with okadaic acid (OA). Thus, under these conditions, L-type channels were unmasked at Bapta- but not at Egta-loaded NMJs. This suggests that the speed, not the capacity, of the calcium chelator was decisive in preventing Ca 2+ -inactivation and facilitating the contribution to neurotransmitter release. At neonatal rat NMJs, N-type VDCCs were involved early during development whereas P/Q-type VDCCs play a main role at all stages of development. Furthermore, P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than N-type VDCCs. This difference could be accounted for by a differential location of these channels at the release site. Neuromuscular transmission in P/Q-type calcium channel knock out ataxic mice jointly depends on both N-type and R-type channels and shows several altered properties including low quantal content. Thus, calcium channels may be recruited to mediate neurotransmitter release with a functional hierarchy where the P/Q channel seems to be the channel most suited to mediate exocytosis at NMJs.     

Molecular evolution and functional divergence of the bestrophin protein family

Background  

Mutations in human bestrophin 1 are associated with at least three autosomal-dominant macular dystrophies including Best disease, adult onset vitelliform macular dystrophy and autosomal dominant vitreo-retinochoroidopathy. The protein is integral to the membrane and is likely involved in Ca²⁺-dependent transport of chloride ions across cellular membranes. Bestrophin 1 together with its three homologues forms a phylogenetically highly conserved family of proteins.     

beta-Adrenergic receptor activation induces internalization of cardiac Cav1.2 channel complexes through a beta-arrestin 1-mediated pathway

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of beta-adrenergic receptors (beta-ARs), which leads to an increase in calcium current (I(Ca-L)) density through cardiac Ca(v)1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I(Ca-L) density and kinetics in heart failure often occur in the absence of changes in Ca(v)1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained beta-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and beta-arrestin 1 (beta-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G(i/o) mediates this response. A peptide that selectively disrupts the interaction between Ca(V)1.2 and beta-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G(i) of the beta-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.     

Ca2+ influx mediates enhanced alpha2-adrenergic contraction in aortas from rats treated with NOS inhibitor

Previously, we reported that aortic segments from rats made hypertensive with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) exhibit enhanced contractile sensitivity to both alpha2-adrenergic receptor (alpha2-AR) stimulation and to KCl-induced depolarization. We hypothesized that increased contractile responses to these agents was due to a change in the common effector L-type voltage-dependent calcium channel (VDCC). In aortic segments from control and L-NNA-treated rats, contraction to the alpha2-AR agonist UK-14304 stimulated Ca2+ influx but released intracellular Ca2+ only in control arteries. UK-14304-induced contraction was blocked by the VDCC antagonist nifedipine in both control and L-NNA aortas but contraction of aortas from L-NNA-treated rats was blocked by lower concentrations. Calcium imaging studies in fura 2-loaded freshly isolated aortic vascular smooth muscle cells also demonstrated UK-14304-stimulated Ca2+ influx sensitive to nifedipine only in cells from L-NNA-treated rats. We conclude that alpha2-AR contraction in the rat aorta is mediated primarily by Ca2+ influx and that L-NNA-induced hypertension increases the dependence of this contraction on VDCCs.