Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

Alternative splicing of the skeletal muscle Ca_V1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant Ca_V1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (Ca_V1.1 null) myotubes reconstituted with Ca_V1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer Ca_V1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer Ca_V1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, Ca_V1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.     

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

The voltage-gated calcium channel Ca_V1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of Ca_V1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire Ca_V1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for Ca_V1.1 and Ca_V1.2 L-type calcium channels. Moreover, in Ca_V1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-Ca_V1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V_1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca_V1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V_1/2, whereas no further shift of V_1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in Ca_V1.1.     

Distinct brain oscillatory responses for the perception and identification of one’s own body from other’s body

The body recognition process includes complex visual processing, the sensation, perception, and distinction stages of the stimulus. This study examined this process by using the time–frequency analysis of EEG signals and analyzed the obtained data by using the event-related oscillations method. This study aimed to examine the oscillatory brain responses and distinguish one’s own body from other’s body. In the present study, 17 young adults were included and the EEGs were recorded with 32 electrodes placed in different locations. Event-related power spectrum and phase-locking analyzes were performed. ITC and ERSP data were analyzed using 2 (condition) × 11 (location) × 2 (hemisphere) ANOVA Design. As we observed a prolonged response in the theta band in the grand averages, we included the time variable in the overall model. As a result, we found that the phase-locking and the event-related power spectrum of the theta response in recognizing one’s own body were higher when compared to the phase-locking and the event-related power spectrum of the theta response in recognizing others’ body (p < 0.05). When the time variable was included, the early theta response was more phase-locked and had a higher power spectrum compared to the late theta response (p < 0.05). As a result of the power spectrum analysis, the condition × hemisphere interaction effect in the beta band was higher in the left hemisphere regarding increased responses in recognizing one’s own body (p < 0.05). As a result of ITC, the main effect of the condition was higher in the recognition of the stimulus of one’s own body (p < 0.05). Finally, the theta oscillator response stood out in distinguishing one’s own body from other’s body. Similarly, the power spectrum in the beta response was higher in the left hemisphere, and this finding is consistent with the literature.     

Distinct Components of Retrograde CaV1.1-RyR1 Coupling Revealed by a Lethal Mutation in RyR1

The molecular basis for excitation-contraction coupling in skeletal muscle is generally thought to involve conformational coupling between the L-type voltage-gated Ca²⁺ channel (Ca_V1.1) and the type 1 ryanodine receptor (RyR1). This coupling is bidirectional; in addition to the orthograde signal from Ca_V1.1 to RyR1 that triggers Ca²⁺ release from the sarcoplasmic reticulum, retrograde signaling from RyR1 to Ca_V1.1 results in increased amplitude and slowed activation kinetics of macroscopic L-type Ca²⁺ current. Orthograde coupling was previously shown to be ablated by a glycine for glutamate substitution at RyR1 position 4242. In this study, we investigated whether the RyR1-E4242G mutation affects retrograde coupling. L-type current in myotubes homozygous for RyR1-E4242G was substantially reduced in amplitude (∼80%) relative to that observed in myotubes from normal control (wild-type and/or heterozygous) myotubes. Analysis of intramembrane gating charge movements and ionic tail current amplitudes indicated that the reduction in current amplitude during step depolarizations was a consequence of both decreased Ca_V1.1 membrane expression (∼50%) and reduced channel P_o (∼55%). In contrast, activation kinetics of the L-type current in RyR1-E4242G myotubes resembled those of normal myotubes, unlike dyspedic (RyR1 null) myotubes in which the L-type currents have markedly accelerated activation kinetics. Exogenous expression of wild-type RyR1 partially restored L-type current density. From these observations, we conclude that mutating residue E4242 affects RyR1 structures critical for retrograde communication with Ca_V1.1. Moreover, we propose that retrograde coupling has two distinct and separable components that are dependent on different structural elements of RyR1.     

A common pathway for charge transport through voltage-sensing domains

Voltage-gated ion channels derive their voltage sensitivity from the movement of specific charged residues in response to a change in transmembrane potential. Several studies on mechanisms of voltage sensing in ion channels support the idea that these gating charges move through a well-defined permeation pathway. This gating pathway in a voltage-gated ion channel can also be mutated to transport free cations, including protons. The recent discovery of proton channels with sequence homology to the voltage-sensing domains suggests that evolution has perhaps exploited the same gating pathway to generate a bona fide voltage-dependent proton transporter. Here we will discuss implications of these findings on the mechanisms underlying charge (and ion) transport by voltage-sensing domains.     

Distinct Biology of Kaposi’s Sarcoma-Associated Herpesvirus from Primary Lesions and Body Cavity Lymphomas

The DNA sequence for Kaposi’s sarcoma-associated herpesvirus was originally detected in Kaposi’s sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi’s sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi’s sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt’s B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi’s sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi’s sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi’s sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi’s sarcoma-associated herpesvirus isolates from primary Kaposi’s sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi’s sarcoma.     

Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains

We propose that the in vivo functions of NM II (non-muscle myosin II) can be divided between those that depend on the N-terminal globular motor domain and those less dependent on motor activity but more dependent on the C-terminal domain. The former, being more dependent on the kinetic properties of NM II to translocate actin filaments, are less amenable to substitution by different NM II isoforms, whereas the in vivo functions of the latter, which involve the structural properties of NM II to cross-link actin filaments, are more amenable to substitution. In light of this hypothesis, we examine the ability of NM II-A, as well as a motor-compromised form of NM II-B, to replace NM II-B and rescue neuroepithelial cell-cell adhesion defects and hydrocephalus in the brain of NM II-B-depleted mice. We also examine the ability of NM II-B as well as chimaeric forms of NM II (II-A head and II-B tail and vice versa) to substitute for NM II-A in cell-cell adhesions in II-A-ablated mice. However, we also show that certain functions, such as neuronal cell migration in the developing brain and vascularization of the mouse embryo and placenta, specifically require NM II-B and II-A respectively.     

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.     

The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases

BLM and WRN, the products of the Bloom’s and Werner’s syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3′→5′ polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded ‘bubble’, as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each ‘arm’ of the 4-way junction is blunt-ended. Surprisingly, a 3′-tailed duplex, a standard substrate for 3′→5′ helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3′-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom’s or Werner’s syndrome.     

Current Therapeutic Options for Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease in the developed world. The increasing life expectancy in the last years has led to an increase in the prevalence of this age-related condition and has posed an important medical and social challenge for developed societies. The mainstays of current therapy for AD rely on the cholinergic hypothesis developed more than 20 years ago. These compounds, known as acetylcholinesterase inhibitors (AChEIs), inhibit the cholinesterases and aim at improving the brain synaptic availability of acetylcholine. These drugs have been approved for the treatment of AD based on pivotal clinical trials showing modest symptomatic benefit on cognitive, behavioral, and global measures. Memantine, an NMDA antagonist, has been recently included as a therapeutic option for AD. Memantine can be combined safely with AChEIs for an additional symptomatic benefit. During the last years our understanding of the mechanisms underlying the pathogenesis of AD has markedly expanded. Several putative neuroprotective drugs are thoroughly investigated and many of them have reached the clinical arena. It can be anticipated that some of these drugs will be able to slow/prevent the progression of this condition in the near future.     

Excitation–Contraction Coupling: Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

The skeletal muscle voltage-gated calcium channel (Ca_V1.1) primarily functions as a voltage sensor for excitation–contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α_1S subunit and the auxiliary α₂δ-1 and γ₁ subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of Ca_V1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α₂δ-1, β₃, and STAC3. When the adult (Ca_V1.1a) and embryonic (Ca_V1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of Ca_V1.1a. Only when we further coexpressed the γ₁ subunit was the current density of Ca_V1.1a, but not that of Ca_V1.1e, reduced by >50%. In addition, γ₁ caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ₁, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in Ca_V1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ₁ subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ₁-dependent reduction of current density, suggesting that structural rearrangements in Ca_V1.1a induced by inclusion of exon 29 may allosterically empower the γ₁ subunit to exert its inhibitory action on Ca_V1.1 calcium currents.     

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.     

Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.