Novel vertebrate homologues detected for two families of mechanosensitive channels.

HYUN JI KIM and MARK S. P. SANSOMStructural Bioinfomatics and Computational Biochemistry Unit, Department of Biochemistry, University of

Oxford, South Parks Road, Oxford, OX1 3QU, UK

*For further information, contact Hyunji Kim Email: hyunji.kim@bioch.ox.ac.uk and Mark Sansom Email: mark.sansom@bioch.ox.ac.uk

1. Genomic scanning of mechanosensitive channels revealed unprecedented vertebrate homologues of Large and Small conductance MS channels.2. It is speculated that Gly and Aln may serve key residues accounting for mechanosensation observed in families of 2 pore potassium channels, Transient Receptor Potential channels and Large/Small mechanosensitive channels.

Phylogenetic Trees with Evolutionary Distance combined.

4A Vertebrate Homologue of Large-conductance MechanoSensitive Channel

2

A Mammalian Homologue of Small-conductance MechanoSensitive Channel

3 3-D Mapping of Sequence Conservation5

(1)Biggin PC, Sansom MSP (2003) Mechanosensitive Channels: Stress Relief. Current Biology 13(5): R183-R185.

(2) Honore E (2007) The neuronal background K2P channels: focus on TREK1. Nature Reviews Neuroscience 8:251- 261

SBCB website, http://sbcb.bioch.ox.ac.uk.

ReferencesConclusions7

6 Mechanosensitivity is traced down to Gly & Aln.

MscL MscS

.

A) b)

C)

A)Sequence alignment of MscL family, colour-coded for two conserved residues, Gly and Aln.B) Pairwise comparison of profile Hidden Markov Models: Font in proportion to emission

probabilities, whilst letters shown per column correspond to symbols emitted from each state.

Chick4_results

Chim

p4_resultsH

uman4_results

Mouse4_results

Zebra1_resultsC

and_gl_pep_resultsEnce_cu_pep_resultsPara_te_pep_resultsYeast_pep_resultsB

aci_su_pep_resultsC

hla_tr_pep_resultsC

ory_gl_pep_resultsH

alo_sa_pep_resultsM

eth_ja_pep_resultsM

eth_th_pep_resultsM

yco_pn_pep_resultsN

ano_eq_pep_resultsPire_sp_pep_resultsPyro_ho_pep_resultsShig_fl1_pep_resultsStap_au3_pep_resultsStre_co_pep_resultsSulf_to_pep_resultsVibr_ch_pep_results

KvAP;S1-S4Bacteriorhodopsin

MscLMscS

Na+_alphaTRP

ChakKvB2

clcAKcsA;S5-S6

nAChR;LBDCa2+_beta1

Ca2+_beta4Kv1.2;S1-S4

ENaC7TM_class1

NBD1SUR1

0

100

200

300

400

500

600

Genomic landscape of ion channels & related proteins

Num

ber of homologues

80 genomes in total, were screened for homologues of 17 ion channels and related proteins. ‘WU-BLAST’ was employed as search-engine, and ‘blastp’ was executed with 6 various amino acid substitution matrices, for enhanced sensitivity, whereas ‘MSPcrunch’ was plugged in our automation-script for removing spurious hits, thus higher specificity.

A Genomic Landscape of Ion Channels1

TMH1

TMH3

A) Sequence alignment of MscS family, Gly (yellow) and Aln (sky blue), as in MscL-TMH1. B) Pairwise comparison of two profile Hidden Markov Models, each representing MscS-TMH3 (upper) and a consensus mechanosensitive TM helix (lower). The latter was co-extracted from two additional protein families, which are two pore potassium channels (K2P) and transient receptor potential (TRP) channels.

A)

A)

B)

B)The vertebrate homologues, detected for MscL and MscS, are predicted to encode three TM helices (Pongo and Dr. Cuthbertson’s). Sequence conservation was measured and mapped onto correspondingcrystal structures, 2oau (MscL) and 2oar (MscS), in Chimera. Degree of conservation is expressed by the thickness of worm-shaped presentation and also by the strength of blue shades.

A)A consensus mechanosensitive motif, detected from 4 protein families, which are MscL, MscS, two pore potassium channel (K2P) and TRP channels. One transmembrane helix (TMH) was extracted from each family, such as TMH1 of MscL, TMH3 of MscS, TMH2 of pore 1 and pore 2 of K2P, and TMH4 of TRP channels.

B) Principal Component Analysis showing co-relationship amongst the above 4 protein families.C) A profile Hidden Markov Model was constructed from the assembled alignment on the left, which was

subsequently visualised by HMM Logos. Notable are strong signals detected for conserved Gly and Aln, despite a very low overall sequence identity across those ion channel families.

MscL

TRPV4NOMPC

K2P

MscS

MscSmouse

MscLchick

MscL MscS

MscL_HAEIN

MscL_PSEFL

MscL_BACSU

MscL_CLOPE

MscL_CHICK

MscL_STAAW

2oar

MscL_MYCTU

MscL_SYNY3

MscL_ECOLI

MscS_ECO57

2oau

MscS_mouse

Q51409_BOR

Q55882_SYN

Q34897_BAC

Q05781_MYC

MscS_EDWIC

MscS_SHIFL

Evolutionary distances were calculated from pairwise sequencecomparison (using PHYLIP), based on the two alignments presented for MscL and MscS. Identical pairs are lined in the central diagonal direction.Each shade of blue increments 0.5 in the range of 0 to 3.5.