V11 Splice varitants in TRP channels

SS 2008 lecture 11

Biological Sequence Analysis1

V11 Splice varitants in TRP channels

Review of lecture V10 .. Introduction of TRP channels

SS 2008 lecture 11


RNA splicing

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

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.

http://upload.wikimedia.org/wikipedia/commons/1/17/Pre-mRNA_to_mRNA.png

SS 2008 lecture 11


alternative splicing

www.wikipedia.org

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.

SS 2008 lecture 11


Arniges et al. J. Biol. Chem. 281, 1580 (2006)

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.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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 1M

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.

SS 2008 lecture 11



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.

SS 2008 lecture 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.

SS 2008 lecture 11



Heteromerization of TRPV4 variants

B, FRET efficiencies

determined between different

TRPV4 variants showed

heterooligomerization only for A

and D proteins.

SS 2008 lecture 11



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.

SS 2008 lecture 11



Summary II

Discovery of three important traits of TRPV4 biogenesis.

1) 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.

SS 2008 lecture 11



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.

SS 2008 lecture 11


Oberwinkler et al. J. Biol. Chem. 280, 22540 (2005)

Next: Identification of TRMP3 splice variants from mouse brain

A, schematic diagram of the mouse Trpm3 gene, comprising 28 exons.

SS 2008 lecture 11



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.

SS 2008 lecture 11



Pore regions of splice variants

D, putative pore regions of TRPM31 and TRPM32 compared with the

corresponding mouse sequences of TRPM6, TRPM7, TRPV5, and TRPV6.

The 12 additional amino acid residues present in TRPM31 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.

SS 2008 lecture 11



TRPM3 functions as cation channel

Heterologous expression of TRPM31 induces outwardly rectifying cation

currents inhibited by intracellular Mg2+.

A,current-voltage relationship of a TRPM31-expressing cell in standard Ringer

or NMDG solution within 60 s after establishing the whole cell patch clamp

configuration.

SS 2008 lecture 11



Permeability for divalent cations

TRPM31 and TRPM32 display large differences in their relative

permeability ratios for divalent cations.

A, comparison of TRPM31 and TRPM32 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.

SS 2008 lecture 11



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).

SS 2008 lecture 11



Effect of extracellular cations

Inhibition of TRPM3-dependent currents

by extracellular cations.

A, comparison of TRPM31 and TRPM32

currents at 80 mV and 80 mV in extra-

cellular solutions containing indicated

amounts of Na+.

Outward currents through TRPM31 are

unaffected by extracellular Na+, whereas

outward currents through TRPM32 are

inhibited in a dose-dependent manner by

these ions.

B, statistical analysis of recordings with

varying concentrations of Na+, K+, Ca2+, and

Mg2+.

TRPM32 is inhibited byall cations tested on the extra-cellular side.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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, TRPM31, preferentially conducts monovalent cation influx, whereas

TRPM32 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.

SS 2008 lecture 11



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.

SS 2008 lecture 11



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 (TRPM32) and 20

(TRPM31) 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 TRPM31 compared with

TRPM32, perhaps simply because of increased electrostatic repulsion.

Block of TRPM3 Channels by Intra- and Extracellular Cations — Both TRPM31

and TRPM32 are regulated by physiological concentrations of intracellular Mg2+,

similar to related members of the TRPM family such as TRPM6 and TRPM7.

V11 Splice varitants in TRP channels

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