Conformational dynamics of helix S6 from Shaker potassium channel: simulation studies

Prolines in transmembrane (TM) alpha-helices are believed to play an important structural and/or functional role in membrane proteins. At a structural level a proline residue distorts alpha-helical structure due to the loss of at least one stabilizing backbone hydrogen bond, and introduces flexibility in the helix that may result in substantial kink and swivel motions about the effective "hinge." At a functional level, for example in Kv channels, it is believed that proline-induced molecular hinges may have a direct role in gating, i.e., the conformational change linked to opening/closing the channel to movement of ions. In this article we study the conformational dynamics of the S6 TM helix from of the Kv channel Shaker, which possesses the motif PVP--a motif that is conserved in Kv channels. We perform multiple molecular dynamics simulations of single S6 helices in a membrane-mimetic environment in order to effectively map the kink-swivel conformational space of the protein, exploiting the ability of multiple simulations to achieve greater sampling. We show that the presence of proline locally perturbs the helix, disrupting local dihedral angles and producing local twist and unwinding in the region of the hinge--an effect that is relaxed with distance from the PVP motif. We furthermore show that motions about the hinge are highly anisotropic, reflecting a preferred region of kink-swivel conformation space that may have implications for the gating process.     

Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na⁺-Ca²⁺ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na⁺-Ca²⁺ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na⁺-Ca²⁺ exchange currents, where pipette Ca²⁺ _o exchanges for bath Na⁺ _i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na⁺ _i affinities and current–voltage relationships, significant differences were observed in their Na⁺ _i- and Ca²⁺ _i-dependent regulatory properties. Both isoforms underwent Na⁺ _i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na⁺ _i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na⁺ _i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na⁺-Ca²⁺ exchange activity by Ca²⁺ _i, but their responses to regulatory Ca²⁺ _i differed markedly. For both isoforms, the application of cytoplasmic Ca²⁺ _i led to a decrease in outward exchange currents. This negative regulation by Ca²⁺ _i is unique to Na⁺-Ca²⁺ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca²⁺ for all other characterized Na⁺-Ca²⁺ exchangers. For CALX1.1, Ca²⁺ _i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca²⁺ _i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca²⁺ _i occurred by a direct mechanism and indirectly through effects on Na⁺ _i-induced inactivation. Our results show that regulatory Ca²⁺ _i decreases Na⁺ _i-induced inactivation of CALX1.2, whereas it stabilizes the Na⁺ _i-induced inactive state of CALX1.1. These effects of Ca²⁺ _i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na⁺-Ca²⁺ exchange proteins.     

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5′ and 3′ splice sites

The genome of the Friend murine leukemia virus (Fr‐MLV) contains a 5′ splice site (5′ss) located at 205 nt and a 3′ss located at 5489 nt. In our previous studies, it was shown that if the HindIII–BglII (879–1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr‐MLV sequence, then cryptic splicing of env‐mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII–BglII fragment were constructed. The vector, in which a 38 bp fragment (1612–1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183–1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI–NdeI (5140–5400 bp) fragment just upstream of the 3′ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140–5400 nt region located just upstream of the 3′ss is required for the splicing function of the 38 nt fragment and its flanking sequences.     

TET‐catalyzed oxidation of intragenic 5‐methylcytosine regulates CTCF‐dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4⁺ T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins.     

Role of nova‐1 in regulating α2N,a novel glycine receptor splice variant,in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT‐PCR, we investigated the regulation of GlyR α‐subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR α2 subunit, α2A and α2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named “α2N.” Examination of the RNA from spinal cords of different‐aged rats showed a dramatic down‐regulation of α2N during prenatal development: α2N mRNA formed a significant portion of the α2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR α2 pre‐mRNA, the neurooncological ventral antigen‐1 (Nova‐1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of α2N, calling into question their involvement in the developmental regulation of α2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova‐1 variants significantly altered the relative level of GlyR α2N, showing that Nova‐1 isoforms can regulate GlyR α2 pre‐mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR α2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova‐1 and brPTB in the developing spinal cord, and suggest that Nova‐1 plays a role in regulating GlyR α2N in developing neurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 156–165, 2002     

Calcium channel γ subunits provide insights into the evolution of this gene family

The γ subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct γ subunits have been described from human and/or mouse. The first identified member of this group of proteins, γ₁, is a component of the L-type calcium channel expressed in skeletal muscle. A second member, γ₂, identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel γ subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian γ subunits suggests the following relationship ((((γ₂, γ₃), (γ₄, γ₈)), (γ₅, γ₇)), (γ₁, γ₆)) that indicates that they evolved from a common ancestral γ subunit via gene duplication. Our analysis reveals that the novel γ subunit γ₆ most closely resembles γ₁ and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other γ subunits. Rat γ subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of γ₁ mRNA and the long isoform of γ₆ mRNA is most robust in skeletal muscle, while γ₆ is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the γ subunits, we propose that γ₁ and γ₆ may share common physiological functions distinct from the other homologous γ subunits.     

Site-specific contacts enable distinct modes of TRPV1 regulation by the potassium channel Kvβ1 subunit

Transient receptor potential vanilloid 1 (TRPV1) channel is a multimodal receptor that is responsible for nociceptive, thermal, and mechanical sensations. However, which biomolecular partners specifically interact with TRPV1 remains to be elucidated. Here, we used cDNA library screening of genes from mouse dorsal root ganglia combined with patch-clamp electrophysiology to identify the voltage-gated potassium channel auxiliary subunit Kvβ1 physically interacting with TRPV1 channel and regulating its function. The interaction was validated in situ using endogenous dorsal root ganglia neurons, as well as a recombinant expression model in HEK 293T cells. The presence of Kvβ1 enhanced the expression stability of TRPV1 channels on the plasma membrane and the nociceptive current density. Surprisingly, Kvβ1 interaction also shifted the temperature threshold for TRPV1 thermal activation. Using site-specific mapping, we further revealed that Kvβ1 interacted with the membrane-distal domain and membrane-proximal domain of TRPV1 to regulate its membrane expression and temperature-activation threshold, respectively. Our data therefore suggest that Kvβ1 is a key element in the TRPV1 signaling complex and exerts dual regulatory effects in a site-specific manner.