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V11 Splice varitants in TRP channels

V11 Splice varitants in TRP channels. Review of lecture V10 .. Introduction of TRP channels. RNA splicing. Splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined.

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V11 Splice varitants in TRP channels

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  1. V11 Splice varitants in TRP channels Review of lecture V10 .. Introduction of TRP channels Biological Sequence Analysis

  2. RNA splicing Splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation. For many eukaryotic introns, splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs), but there are also self-splicing introns. Simple illustration of exons and introns in pre-mRNA and the formation of mature mRNA by splicing. The UTRs are non-coding parts of exons at the ends of the mRNA. www.wikipedia.org Biological Sequence Analysis

  3. alternative splicing Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript, the pre-mRNA, are separated and reconnected so as to produce alternative RNA arrangements. Via translation, these then give different (isoform) proteins. In this way, alternative splicing uses genetic expression to facilitate the synthesis of a greater variety of proteins. Alternative splicing is of great importance to genetics - it invalidates the old "one-gene-one-protein" hypothesis. External information is needed in order to decide which polypeptide is produced, given a DNA sequence and pre-mRNA. The amount of alternative splicing is comparable, with no large differences between humans and other animals. The "record-holder" for alternative splicing is a Drosophila gene called Dscam, which has 38 016 splice variants. www.wikipedia.org Biological Sequence Analysis

  4. TRPV4 channels The non-selective cation channel TRPV4 is a member of the transient receptor potential (TRP) family of channels. TRPV4 shows multiple modes of activation and regulatory sites, enabling it to respond to various stimuli, including osmotic cell swelling, mechanical stress, heat, acidic pH, endogenous ligands, high viscous solutions, and synthetic agonists such as 4-phorbol 12,13-didecanoate. TRPV4 mRNA is expressed in a broad range of tissues, although functional tests have only been carried out in a few: endothelial, epithelial, smooth muscle, keratinocytes, and DRG neurons. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  5. Topology of TRPV4 channels The general topology of a TRP subunit consists of 6 predicted TM domains with a putative pore loop between TMD5 and TMD6 and intracellular N- and C-terminal regions of variable length, the former containing multiple ankyrin (ANK) repeats in the TRPC, TRPA, TRPN, and TRPV subfamilies. ANK repeats are modular protein interaction domains, each composed by 33 amino acids with a highly conserved helix turn helix motif that determines ist interaction properties. Functional TRP channels are supposed to result following the assembly of 4 TRP subunits. The rules governing subunit assembly and the protein domains implied in this oligomerization process are just starting to emerge and may involve the cytosolic N-terminal region, the ANK domains, transmembrane domains, and the cytoplasmic C terminus. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  6. Cloning of TRPV4 variants from human airway epithelial cells • A reverse transcriptase-PCR-based cloning process identified 5 variants of the TRPV4 channel in human tracheal epithelial cells. • 2 of the cloned cDNAs corresponded to the already described • TRPV4 isoform A (fulllength cDNA) and • TRPV4 isoform B (lacking exon number 7, 384–444 amino acids). • We also identified 3 new splice variants affecting the cytoplasmic N-terminal region. • TRPV4-C lacks exon 5 (237–284 amino acids), • TRPV4-D presents a short deletion inside exon 2 (27–61 amino acids), and TRPV4-E (237–284 and 384–444 amino acids) is produced by a double alternative splicing lacking exons 5 and 7. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  7. Different splice variants of TRPV4 A, schematic diagram showing the intracellular N-terminal region of the human TRPV4 channel (amino acids 1–471). Exons and the corresponding amino acids lost in each TRPV4 isoform are indicated by numbers. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  8. Functional analysis of TRPV4 variants: intracellular [Ca2+] The TRPV4-A channel responds to a wide variety of stimuli. Here, HeLa cells were transiently transfected and intracellular Calcium concentration was determined via Fura-2 ratios as reponse to 3 well known activators of TRPV4-A: 30% hypotonic solution, 1 M 4-PDD, or 10 M arachidonic acid  Only TRPV4-A and TRPV4-D show channel activity. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  9. TRPV4-A and D produce functional channels TRPV4-A and TRPV4-D isoforms produce functional channels with similar properties when expressed in HEK-293 cells. A, current traces obtained from TRPV4-A and TRPV4-D-expressing HEK-293 cells at the indicated voltages in the presence of 1M 4-PDD. Dashed lines indicate the zero current level. B, I–V relationship of 4-PDD-activated TRPV4-A (open circle) and TRPV4-D (closed circle) channels in inside-out patches. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  10. Retention in ER Co-localization experiments (not shown): TRPV4-B, C and E are trapped in the ER and not translocated to the plasma membrane. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  11. Homomerization of TRPV4 variants FRET efficiencies determined between identical CFP- and YFP-fused TRPV4 variants (A–E) transiently cotransfected in HEK-293 cells. High FRET efficiencies corresponding to homomultimer formation could only be demonstrated for TRPV4-A and TRPV4-D variants. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  12. Heteromerization of TRPV4 variants B, FRET efficiencies determined between different TRPV4 variants showed heterooligomerization only for A and D proteins. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  13. Summary This study of oligomerization, localization, and channel activity of human TRPV4 splice variant identified the N-terminal ANK repeats as key molecular determinants of subunit assembly and subsequent processing of the assembled channel. Five TRPV4 variants (TRPV4-A–E) cloned from human airway epithelial cells were grouped into two classes: group I: TRPV4-A and TRPV4-D group II: TRPV4-B, TRPV4-C, and TRPV4-E. Group I variants are correctly processed and targeted to the plasma membrane where they form functional channels with similar electrophysiological properties. Variants from group II, which are lacking parts of the ANK domains are unable to oligomerize and were retained intracellularly, in the ER. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  14. Summary II • Discovery of three important traits of TRPV4 biogenesis. • Glycosylation of TRPV4 channel involves ER to Golgi transport with the corresponding change in the N-linked oligosaccharides from the high mannose type characteristic of the ER to the complex type characteristic of the Golgi apparatus, without apparent O-glycosylation. • 2) TRPV4-A subunits oligomerize in the ER. • 3) Impaired subunit assembly of type II variants is because of the lack of N-terminal ANK domains and causes protein retention in the ER. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  15. Summary III Ion channel functional diversity is greatly enlarged by both the presence of splice variants and heteromerization of different pore-forming and regulatory subunits. Alternative splicing is a major contributor to protein diversity. Within the TRP family of ion channels several splice variants have been identified, some of them resulting in lack of responses to typical stimuli, others modifying the pore properties, and those exerting dominant negative effects. Group II TRPV4 splice variants have been identified in two unrelated, human airway epithelial cell lines. Considering the relevance of TRPV4 channels in epithelial physiology, a change in the expressed ratio of group I to group II variants, favoring the later, may modify normal epithelial functioning. Splicing can be regulated by several stressing stimuli including pH, osmotic, and temperature shocks, all of them being also activating stimuli of the TRPV4. Arniges et al. J. Biol. Chem. 281, 1580 (2006) Biological Sequence Analysis

  16. Next: Identification of TRMP3 splice variants from mouse brain A, schematic diagram of the mouse Trpm3 gene, comprising 28 exons. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  17. TRPM3 splice variants C, schematic presentation of TRPM3 with transmembrane domains 1–6, coiled coil region (cc), and TRP homology domain (Trp). Novel mouse TRPM3 protein variants shown as thick black lines are compared with the human variants hTRPM3a–f and hTRPM31325. The numbers of amino acid residues of each variant are indicated in parentheses. Starting from residue 156, mouse and human TRPM3 have 97% sequence identity. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  18. Pore regions of splice variants D, putative pore regions of TRPM31 and TRPM32 compared with the corresponding mouse sequences of TRPM6, TRPM7, TRPV5, and TRPV6. The 12 additional amino acid residues present in TRPM31 are indicated. Identical residues are boxed in black, conserved in gray. An aspartate residue that determines Ca2+ permeation of the TRPV5/TRPV6 pore is marked by an asterisk. Residues proposed to build the selectivity filter of TRPV6 are underlined. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  19. TRPM3 functions as cation channel Heterologous expression of TRPM31 induces outwardly rectifying cation currents inhibited by intracellular Mg2+. A,current-voltage relationship of a TRPM31-expressing cell in standard Ringer or NMDG solution within 60 s after establishing the whole cell patch clamp configuration. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  20. Permeability for divalent cations TRPM31 and TRPM32 display large differences in their relative permeability ratios for divalent cations. A, comparison of TRPM31 and TRPM32 currents at 80 mV and 80 mV in extracellular solutions containing indicated amounts of Ca2+. B, reversal potential during the experiment shown in panel A. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  21. Identification of TRMP3 variants from mouse brain C and D, statistical analysis of reversal potential measurements in experiments similar to that shown in panel B during the application of solutions containing the indicated concentration of Ca2+ (C) or Mg2+ (D) as the only permeable ion. Continuous thin lines show the expected reversal potential calculated from Goldman-Hodgkin-Katz theory for the indicated relative permeability ratios. Each point represents the mean of 3–15 independent measurements (at a divalent concentration of 10 mM p < 0.001, otherwise at least p < 0.05). Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  22. Effect of extracellular cations Inhibition of TRPM3-dependent currents by extracellular cations. A, comparison of TRPM31 and TRPM32 currents at 80 mV and 80 mV in extra-cellular solutions containing indicated amounts of Na+. Outward currents through TRPM31 are unaffected by extracellular Na+, whereas outward currents through TRPM32 are inhibited in a dose-dependent manner by these ions. B, statistical analysis of recordings with varying concentrations of Na+, K+, Ca2+, and Mg2+. • TRPM32 is inhibited by all cations tested on the extra- cellular side. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  23. Summary Alternative Splicing Switches the Ion Selectivity of TRPM3 Channels— The selectivity of ion channels is thought to be determined by the geometry and charge distribution of the selectivity filter, usually envisioned as the narrowest part of the channel pore. Typically, all members of an ion channel family, such as voltage-gated Na+, K+, or Ca2+ channels, share common ionic selectivities. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  24. Summary II The TRP family of ion channels is already somewhat unusual in this respect as it encompasses members with quite diverging cationic selectivity profiles. The Trpm3 gene adds extra complexity to this picture, because two channels can be expressed from this gene with entirely different ionic selectivities. One channel, TRPM31, preferentially conducts monovalent cation influx, whereas TRPM32 strongly favors divalent entry. In vivo, such a change in ionic selectivity must be expected to have considerable consequences for the function of the channel and the physiology of the cell that expresses it. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  25. Summary III Locating the Ion-conducting Pore in TRPM Channels—The switch of ionic selectivity in TRPM3 variants is brought about by removing a short stretch of 12 amino acid residues and exchanging 1 further residue within the linker domain between the presumed fifth and sixth transmembrane regions (Fig. 1B). The differences in ion selectivity seen for the TRPM3 splice variants strongly indicate that this linker domain constitutes the pore of TRPM3. Although this domain could already be suspected to be the ion-conducting pore, due to direct evidence obtained for TRPV1, TRPV4, TRPV5, and TRPV6 channels, this prediction has not been confirmed up to now for any member of the TRPM subfamily. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

  26. Summary IV Compared with the presumed pore regions of other members of the TRP family, the pore loop of TRPM3 is considerably longer by 8 (TRPM32) and 20 (TRPM31) additional amino acid residues. The domains that build the proposed selectivity filter of the Ca2+-selective TRPV5/V6 channels are conserved in TRPM3 proteins. The splicing within the TRPM3 channel pore introduces additional, positively charged amino acid residues into this domain. This might decrease the Ca2+ permeability of TRPM31 compared with TRPM32, perhaps simply because of increased electrostatic repulsion. Block of TRPM3 Channels by Intra- and Extracellular Cations — Both TRPM31 and TRPM32 are regulated by physiological concentrations of intracellular Mg2+, similar to related members of the TRPM family such as TRPM6 and TRPM7. Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005) Biological Sequence Analysis

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