The

cys-loop

ligand-gated

ion

channel

gene

superfamily

of

the

nematode

Caenorhabditis

elegans

The

nematode

Caenorhabditis

elegans

possesses

the

most

extensive

known

superfamily

of

cys-loop

ligand-gated

ion

channels

-LRB-

cys-loop

LGICs

-RRB-

consisting

of

subunit-encoding

genes

Less

than

half

of

these

genes

have

been

functionally

characterised

which

include

cation-permeable

channels

gated

by

acetylcholine

-LRB-

ACh

-RRB-

and

γ-aminobutyric

acid

-LRB-

GABA

-RRB-

as

well

as

anion-selective

channels

gated

by

ACh

GABA

glutamate

and

serotonin

Following

the

guidelines

set

for

genetic

nomenclature

for

C.

elegans

we

have

designated

unnamed

subunits

as

lgc

genes

-LRB-

ligand-gated

ion

channels

of

the

cys-loop

superfamily

-RRB-

Phylogenetic

analysis

shows

that

several

of

these

lgc

subunits

form

distinct

groups

which

may

represent

novel

cys-loop

LGIC

subtypes

Introduction

Members

of

the

cysteine-loop

ligand-gated

ion

channel

-LRB-

cys-loop

LGIC

-RRB-

superfamily

are

neurotransmitter

receptors

that

mediate

synaptic

transmission

in

vertebrates

and

invertebrates

Cys-loop

LGICs

are

made

up

of

five

homologous

subunits

arranged

around

a

central

ion

channel

-LRB-

Sine

and

Engel

-RRB-

The

characteristic

cys-loop

motif

is

situated

in

the

N-terminal

extracellular

ligand-binding

region

of

each

subunit

and

consists

of

two

disulphide-bond

forming

cysteines

separated

by

amino

acid

residues

several

of

which

are

highly

conserved

to

form

a

signature

sequence

-LRB-

Gene

Ontology

ID

GO

-RRB-

The

cys-loop

plays

roles

in

receptor

assembly

-LRB-

Green

and

Wanamaker

-RRB-

and

gating

of

the

ion

channel

-LRB-

Grutter

et

al.

-RRB-

The

subunit

composition

determines

the

functional

and

pharmacological

properties

of

the

cys-loop

LGIC

thus

receptor

diversity

is

generated

by

multiple

subunit-encoding

genes

For

instance

the

human

cys-loop

LGIC

superfamily

consists

of

genes

encoding

cation-permeable

nicotinic

acetylcholine

receptors

-LRB-

nAChRs

-RRB-

-LRB-

Kalamida

et

al.

-RRB-

cation-permeable

serotonin

or

type

-LRB-

-RRB-

receptors

-LRB-

Reeves

and

Lummis

-RRB-

anion-permeable

γ-aminobutyric

acid

-LRB-

GABA

-RRB-

type

A

and

C

-LRB-

GABAA

and

GABAC

-RRB-

receptors

-LRB-

Bormann

Darlison

et

al.

-RRB-

and

anion-permeable

glycine

receptors

-LRB-

Kirsch

-RRB-

As

demonstrated

in

the

fruit

fly

-LRB-

Drosophila

melanogaster

-RRB-

honey

bee

-LRB-

Apis

mellifera

-RRB-

and

red

flour

beetle

-LRB-

Tribolium

castaneum

-RRB-

insect

cys-loop

LGIC

superfamilies

are

more

compact

consisting

of

just

over

subunits

-LRB-

Jones

and

Sattelle

Jones

and

Sattelle

Littleton

and

Ganetzky

-RRB-

Approximately

of

these

genes

are

known

to

encode

cation-permeable

nAChRs

-LRB-

Sattelle

et

al.

-RRB-

anion

and

possibly

cation

channels

gated

by

GABA

-LRB-

Buckingham

et

al.

-RRB-

glutamate-gated

anion

channels

-LRB-

Vassilatis

et

al.

-RRB-

and

histamine-gated

anion

channels

-LRB-

Gisselmann

et

al.

Zheng

et

al.

-RRB-

The

largest

known

cys-loop

LGIC

superfamily

belongs

to

the

simple

nematode

Caenorhabditis

elegans

where

an

early

analysis

of

its

completed

genome

revealed

ligand-gated

ion

channel

genes

-LRB-

Bargmann

-RRB-

In

common

with

vertebrates

members

of

this

large

cys-loop

LGIC

superfamily

include

cation-permeable

nAChRs

-LRB-

Jones

and

Sattelle

-RRB-

and

anion-permeable

GABA

receptors

-LRB-

Schuske

et

al.

-RRB-

As

is

the

case

for

insects

C.

elegans

has

glutamate-gated

anion

channels

-LRB-

Wolstenholme

and

Rogers

-RRB-

but

so

far

no

nematode

histamine-gated

ion

channels

have

been

found

However

C.

elegans

possesses

cys-loop

LGIC

receptors

not

identified

in

vertebrates

and

insects

which

include

anion

channels

gated

by

acetylcholine

-LRB-

Putrenko

et

al.

-RRB-

and

serotonin

-LRB-

Ranganathan

et

al.

-RRB-

The

considerable

diversity

of

receptor

subtypes

in

C.

elegans

may

be

broadened

further

as

many

cys-loop

LGIC

subunits

have

yet

to

be

characterised

Here

we

present

an

update

on

the

genes

present

in

the

C.

elegans

cys-loop

LGIC

superfamily

Methods

All

C.

elegans

protein

sequences

bearing

motifs

particular

to

cys-loop

LGICs

were

assembled

from

WormBase

-LRB-

release

Dec

-RRB-

These

motifs

and

their

source

databases

are

neurotransmitter-gated

ion-channel

ligand-binding

INTERPRO

neurotransmitter-gated

ion-channel

INTERPRO

neurotransmitter-gated

ion-channel

ligand

binding

domain

PFAM

neurotransmitter-gated

ion-channel

transmembrane

region

PFAM

and

ligand-gated

ion

channel

Cys-loop

and

sequences

were

identified

from

an

alignment

of

protein

sequences

of

whole

subunits

constructed

with

ClustalX

-LRB-

Thompson

et

al.

-RRB-

using

the

slow-accurate

mode

with

a

gap

opening

penalty

of

and

a

gap

extension

penalty

of

as

well

as

applying

the

Gonnet

protein

weight

matrix

-LRB-

Benner

et

al.

-RRB-

The

protein

alignment

was

viewed

using

GeneDoc

-LRB-

-RRB-

The

neighbour-joining

method

-LRB-

Saitou

and

Nei

-RRB-

available

with

the

ClustalX

program

was

used

to

construct

a

phylogenetic

tree

which

was

then

displayed

using

the

TreeView

application

-LRB-

Page

-RRB-

Results

and

discussion

Our

analysis

of

the

C.

elegans

genome

revealed

the

largest

cys-loop

LGIC

superfamily

so

far

characterised

consisting

of

genes

As

we

have

previously

reported

the

C.

elegans

nAChR

and

orphan

subunits

-LRB-

Brown

et

al.

Jones

et

al.

Jones

and

Sattelle

Mongan

et

al.

-RRB-

we

concentrate

here

on

the

remaining

cys-loop

LGIC

genes

For

unnamed

subunit

genes

we

continued

to

use

the

nomenclature

first

used

for

orphan

nAChR

subunits

-LRB-

Jones

et

al.

-RRB-

Thus

genes

were

designated

lgc

for

ligand-gated

ion

channel

of

the

cys-loop

LGIC

superfamily

-LRB-

Table

-RRB-

As

shown

in

Fig.

the

C.

elegans

cys-loop

LGIC

subunits

were

grouped

according

to

sequence

homology

As

with

the

C.

elegans

nAChR

subunits

-LRB-

Mongan

et

al.

-RRB-

each

group

was

named

if

possible

after

a

well

characterised

subunit

For

a

detailed

comparison

of

C.

elegans

cys-loop

LGIC

subunits

with

those

of

other

organisms

see

-LRB-

Dent

-RRB-

Table

elegans

cys-loop

LGIC

subunits

that

have

been

given

names

in

this

report

Amino

acid

residues

preceding

that

are

important

for

determining

the

charge

selectivity

of

the

ion

channel

-LRB-

Jensen

et

al.

-RRB-

are

given

The

cys-loop

sequence

is

shown

with

the

cysteine

residues

highlighted

in

black

shading

while

gray

shading

indicates

conserved

residues

The

presence

of

a

putative

second

cys-loop

-LRB-

Dent

-RRB-

is

also

notedFig

showing

the

cys-loop

LGIC

gene

superfamily

of

C.

elegans

This

figure

focuses

on

cys-loop

LGIC

subunits

other

than

nAChRs

although

representative

subunits

of

the

nAChR

groups

are

shown

For

the

complete

C.

elegans

nAChR

gene

family

see

-LRB-

Jones

et

al.

-RRB-

Based

on

protein

sequence

homology

the

subunits

are

divided

into

eight

groups

with

three

highly

divergent

subunits

and

not

belonging

to

any

group

Ungrouped

subunits

Three

subunits

and

were

not

placed

into

any

groups

as

they

are

highly

divergent

-LRB-

Fig.

-RRB-

only

showing

up

to

and

identity

respectively

with

other

C.

elegans

human

and

D.

melanogaster

cys-loop

LGICs

These

subunits

have

not

been

functionally

characterised

so

it

is

unknown

what

ligands

act

on

them

The

sequence

of

and

a

close

homologue

in

the

parasitic

nematode

Dirofilaria

immitis

has

been

previously

described

where

it

has

been

noted

that

even

though

the

subunit

possesses

features

common

to

cys-loop

LGICs

such

as

conserved

residues

in

the

N-terminal

extracellular

region

and

four

transmembrane

domains

the

cys-loop

is

absent

-LRB-

Yates

and

Wolstenholme

-RRB-

Interestingly

a

bacterial

proton-gated

ion

channel

-LRB-

Glvi

-RRB-

resembling

cys-loop

LGICs

but

also

lacking

the

cys-loop

has

been

recently

reported

-LRB-

Bocquet

et

al.

Tasneem

et

al.

-RRB-

may

thus

represent

more

ancestral

members

of

the

cys-loop

LGIC

superfamily

Unlike

Glvi

possesses

two

cysteine

residues

which

are

conserved

in

glutamate

histamine

and

vertebrate

glycine-gated

anion

channels

-LRB-

Dent

Yates

and

Wolstenholme

-RRB-

Known

as

the

second

cys-loop

these

two

cysteines

flank

loop

C

which

is

a

region

that

contributes

to

ligand

binding

-LRB-

Corringer

et

al.

-RRB-

The

Group

This

group

consists

of

only

two

subunits

one

of

which

is

a

GABA-gated

cation

channel

that

mediates

enteric

muscle

contraction

-LRB-

Beg

and

Jorgensen

-RRB-

While

overall

resembles

more

closely

anion

permeable

cys-loop

LGICs

such

as

as

opposed

to

cation-permeable

nAChRs

it

lacks

the

PAR

motif

preceding

the

second

transmembrane

domain

-LRB-

-RRB-

which

is

important

for

anion

selectivity

-LRB-

Jensen

et

al.

-RRB-

Instead

it

possesses

the

residues

ETE

where

the

presence

of

glutamic

acid

residues

has

been

implicated

in

determining

cation

selectivity

-LRB-

Jensen

et

al.

Wotring

and

Weiss

-RRB-

The

other

subunit

in

this

group

also

has

a

glutamate

residue

preceding

-LRB-

Table

-RRB-

and

thus

may

be

a

cation

channel

The

Group

This

group

consists

of

six

subunits

-LRB-

Fig.

-RRB-

that

make

up

glutamate-gated

chloride

channels

that

are

targeted

by

the

avermectin

and

milbemycin

anthelmintics

-LRB-

Wolstenholme

and

Rogers

-RRB-

Examples

of

GluCl

function

include

and

in

the

inhibition

of

pharyngeal

pumping

-LRB-

Dent

et

al.

Laughton

et

al.

Pemberton

et

al.

-RRB-

and

and

in

regulating

locomotion

-LRB-

Cook

et

al.

-RRB-

Recently

it

has

been

shown

plays

a

role

in

olfactory

behaviour

-LRB-

Chalasani

et

al.

-RRB-

The

Group

Products

of

the

gene

form

a

GABA-gated

anion

channel

that

mediates

body

muscle

inhibition

during

locomotion

-LRB-

Bamber

et

al.

Richmond

and

Jorgensen

-RRB-

There

are

four

other

subunits

included

in

this

group

-LRB-

Fig.

-RRB-

With

regard

to

other

species

members

of

the

group

are

clearly

most

closely

related

to

mammalian

and

insect

GABA-gated

anion

channels

-LRB-

Dent

-RRB-

forms

functional

GABA

receptors

when

coexpressed

in

Xenopus

laevis

oocytes

with

either

or

which

are

putative

GABA

receptor

subunits

from

the

parasitic

nematode

Haemonchus

contortus

-LRB-

Feng

et

al.

-RRB-

The

remaining

subunits

have

yet

to

be

characterised

but

it

is

worth

noting

that

possesses

the

amino

acids

AER

instead

of

PAR

before

-LRB-

Table

-RRB-

The

presence

of

a

glutamic

acid

residue

at

this

position

and

the

absence

of

a

proline

may

result

in

a

cation-selective

channel

-LRB-

Jensen

et

al.

-RRB-

The

Group

This

group

includes

six

subunits

-LRB-

Fig.

-RRB-

the

functional

characterisation

of

which

has

so

far

not

been

published

The

neurotransmitters/ligands

to

which

group

subunits

respond

to

can

not

be

easily

inferred

through

phylogenetic

analysis

For

example

when

comparing

with

human

cys-loop

LGICs

they

bear

closest

resemblance

to

glycine

α

subunits

with

approximately

identity

while

is

slightly

more

similar

to

histamine-gated

anion

channels

with

identity

when

comparing

with

insect

cys-loop

LGICs

Members

of

the

group

have

been

previously

denoted

as

I

subunits

which

most

closely

resemble

insect

histamine-gated

anion

channels

-LRB-

Dent

-RRB-

It

would

be

of

interest

to

determine

if

members

of

the

group

respond

to

glycine

histamine

or

other

ligands

not

so

far

known

to

act

on

C.

elegans

cys-loop

LGICs

The

subunits

of

the

group

are

likely

to

be

anion-selective

as

they

all

possess

the

PAR

motif

before

-LRB-

Table

-RRB-

which

is

important

for

anion

selectivity

-LRB-

Jensen

et

al.

-RRB-

The

only

exception

is

which

instead

has

the

amino

acid

residues

PGR

With

the

proline

still

present

and

an

absence

of

an

acidic

residue

-LRB-

particularly

glutamic

acid

-RRB-

in

this

region

it

is

likely

that

will

also

be

anion

selective

The

Group

This

group

consists

of

three

subunits

-LRB-

Fig.

-RRB-

the

functional

characterisation

of

which

has

so

far

not

been

published

As

is

the

case

for

members

of

the

group

and

most

closely

resemble

glycine

α

subunits

and

histamine-gated

anion

channels

when

compared

with

human

and

insect

cys-loop

LGICs

respectively

However

unlike

the

group

the

group

subunits

are

likely

to

be

cation

selective

as

they

lack

the

PAR

motif

before

-LRB-

Table

-RRB-

which

is

important

for

anion

selectivity

-LRB-

Jensen

et

al.

-RRB-

In

particular

and

possess

glutamic

acid

instead

of

the

proline

residue

which

is

likely

to

result

in

a

cation-selective

channel

-LRB-

Wotring

and

Weiss

-RRB-

While

shows

notable

sequence

identity

with

other

cys-loop

LGIC

subunits

-LRB-

up

to

with

-RRB-

it

lacks

the

cys-loop

-LRB-

Table

-RRB-

thus

it

may

represent

an

ancestral

cys-loop

LGIC

subunit

as

has

been

noted

for

The

Group

This

group

includes

and

which

are

acetylcholine-gated

anion

channels

-LRB-

Putrenko

et

al.

-RRB-

Acetylcholine-gated

anion

channels

have

also

been

characterised

from

the

snail

Lymnae

stagnalis

which

appear

to

have

evolved

from

cation

channels

through

amino

acid

substitutions

in

the

ion

channel

pore

-LRB-

van

Nierop

et

al.

-RRB-

In

contrast

acetylcholine-gated

anion

channels

of

the

group

may

have

arisen

from

substitutions

in

the

ligand-binding

domain

of

anion

channels

-LRB-

Putrenko

et

al.

van

Nierop

et

al.

-RRB-

The

Group

modulates

locomotory

behaviour

and

is

the

only

known

invertebrate

cys-loop

LGIC

to

be

gated

by

serotonin

-LRB-

Ranganathan

et

al.

-RRB-

Unlike

mammals

which

possess

cation-permeable

serotonin

receptors

-LRB-

Reeves

and

Lummis

-RRB-

is

anion

selective

One

other

subunit

is

closely

related

to

-LRB-

Fig.

-RRB-

The

Group

This

group

includes

six

subunits

-LRB-

Fig.

-RRB-

the

functional

characterisation

of

which

has

so

far

not

been

published

All

six

subunits

are

likely

to

form

anion-selective

channels

as

they

have

the

PAR

amino

acid

motif

before

-LRB-

Table

-RRB-

-LRB-

Jensen

et

al.

-RRB-

When

comparing

with

human

cys-loop

LGICs

they

bear

closest

resemblance

to

GABA

α

γ

and

ε

subunits

with

approximately

identity

while

they

most

closely

resemble

histamine-gated

chloride

channels

-LRB-

identity

-RRB-

when

comparing

with

insect

cys-loop

LGICs

As

with

the

group

it

would

be

of

interest

to

determine

if

the

group

consists

of

ion

channels

responding

to

ligands

not

so

far

known

to

act

on

C.

elegans

cys-loop

LGICs

Cys-loop

LGIC

superfamilies

of

other

nematode

species

Genome

sequencing

projects

are

allowing

the

comparison

of

cys-loop

LGIC

superfamilies

from

different

nematode

species

For

instance

Caenorhabditis

briggsae

which

is

seemingly

identical

to

C.

elegans

-LRB-

Gupta

et

al.

Stein

et

al.

-RRB-

also

has

an

extensive

cys-loop

LGIC

gene

superfamily

although

there

are

a

few

exceptions

where

there

appears

to

be

gene

expansions

in

C.

elegans

mainly

within

the

nAChR

orphan

group

For

example

and

in

C.

elegans

-LRB-

Jones

et

al.

-RRB-

may

be

paralogues

resulting

from

gene

duplication

in

the

elegans

species

lineage

since

only

one

homologue

-LRB-

-RRB-

is

observed

in

C.

briggsae

In

line

with

this

and

are

situated

close

together

within

kb

in

the

C.

elegans

genome

which

may

reflect

a

recent

gene

duplication

event

Likewise

and

as

well

as

and

have

single

homologues

in

C.

briggsae

and

respectively

Six

C.

elegans

cys-loop

LGIC

subunits

to

are

tightly

clustered

within

kb

of

the

cosmid

whereas

in

C.

briggsae

there

are

seven

similar

genes

-LRB-

and

-RRB-

all

of

which

are

not

clustered

together

Outside

of

the

nAChR

orphan

group

and

may

be

paralogues

arising

in

the

elegans

lineage

as

there

is

only

a

single

homologue

-LRB-

-RRB-

in

C.

briggsae

The

cys-loop

LGIC

gene

families

of

the

parasitic

nematodes

Brugia

malyai

and

Trichinella

spiralis

are

much

smaller

than

those

of

C.

elegans

and

C.

briggsae

consisting

of

and

subunits

respectively

-LRB-

Williamson

et

al.

-RRB-

The

cys-loop

LGIC

subunits

of

both

parasites

have

clear

orthologous

relationships

with

those

of

C.

elegans

with

one

exception

a

nAChR

subunit

-LRB-

-RRB-

which

so

far

appears

particular

to

B.

malayi

As

discussed

by

Williamson

and

colleagues

the

striking

difference

in

the

cys-loop

LGIC

gene

family

sizes

may

reflect

a

free-living

versus

a

parasitic

lifestyle

Thus

the

large

complement

of

subunits

in

C.

elegans

and

C.

briggsae

may

be

required

for

a

small

nervous

system

to

be

able

to

respond

to

many

environmental

cues

which

may

be

more

restricted

in

the

specific

environmental

niche

occupied

by

parasitic

nematodes

This

shows

that

the

extensive

cys-loop

LGIC

gene

superfamily

observed

for

C.

elegans

is

not

a

feature

of

all

nematodes

O O O B I O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O B O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

caenorhabditis elegans
ion channel
acetylcholine
gaba
glutamate
serotonin