Group Title: Genome biology
Title: Comparison of K+-channel genes within the genomes of Anopheles gambiae and Drosophila melanogaster
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Title: Comparison of K+-channel genes within the genomes of Anopheles gambiae and Drosophila melanogaster
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Language: English
Creator: McCormack,Thomas
Publisher: Genome Biology
Publication Date: 2003
Abstract: BACKGROUND:Potassium channels are the largest and most diverse type of ion channel found in nature. The completion of the sequencing of the genomes of Drosophila melanogaster and Anopheles gambiae, which belong to the same order, the Diptera, allows us to compare and contrast K+-channel genes and gene families present within the genomes of two dipterans.RESULTS:This study identifies at least eight voltage-gated K+-channel genes in Anopheles, as well as three Slo-family, three Eag-family and six inward rectifier K+-channel genes. The genomic organization of K+-channel genes from Drosophila and Anopheles is well conserved. The sequence identity of the most similar K+-channel gene products between these two species ranges from 42% to 98%, with a mean value of 85%. Although most K+-channel genes in Drosophila and Anopheles are present in a 1:1 ratio, Anopheles has more genes in three K+-channel types, namely KQT, Kv3, and inward rectifier channels. Microsynteny between the genes flanking K+-channel genes in Drosophila and Anopheles was seldom observed; however, most of the K+-channel genes are indeed located at positions which a previous genome-wide comparison has designated as homologous chromosomal regions.CONCLUSIONS:The Anopheles genome encodes more voltage-gated and inward rectifier K+-channel genes than that of Drosophila. Despite the conservation of intron-exon boundaries, orthologs of genes flanking K+-channel genes in Drosophila are generally not found adjacent to the Anopheles K+-channel orthologs, suggesting that extensive translocation of genes has occurred since the divergence of these two organisms.
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Comparison of K+-channel genes within the genomes of Anopheles

gambiae and Drosophila melanogaster
Thomas J McCormack

Address: Department of Chemistry, University of Florida, Gainesville, FL 32611, USA. Present address: Department of Pharmacology and
Therapeutics, University of Florida, Gainesville, FL 32610-0267, USA. E-mail:

Published: 20 August 2003
Genome Biology 2003, 4:R58
The electronic version of this article is the complete one and can be
found online at

Received: I I March 2003
Revised: I I June 2003
Accepted: 24 July 2003

2003 McCormack; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.


Background: Potassium channels are the largest and most diverse type of ion channel found in
nature. The completion of the sequencing of the genomes of Drosophila melanogaster and Anopheles
gambiae, which belong to the same order, the Diptera, allows us to compare and contrast K+-
channel genes and gene families present within the genomes of two dipterans.

Results: This study identifies at least eight voltage-gated K+-channel genes in Anopheles, as well as
three Slo-family, three Eag-family and six inward rectifier K+-channel genes. The genomic
organization of K-channel genes from Drosophila and Anopheles is well conserved. The sequence
identity of the most similar K+-channel gene products between these two species ranges from 42%
to 98%, with a mean value of 85%. Although most K-channel genes in Drosophila and Anopheles are
present in a 1:1 ratio, Anopheles has more genes in three K-channel types, namely KQT, Kv3, and
inward rectifier channels. Microsynteny between the genes flanking K+-channel genes in Drosophila
and Anopheles was seldom observed; however, most of the K+-channel genes are indeed located at
positions which a previous genome-wide comparison has designated as homologous chromosomal

Conclusions: The Anopheles genome encodes more voltage-gated and inward rectifier K-channel
genes than that of Drosophila. Despite the conservation of intron-exon boundaries, orthologs of
genes flanking K+-channel genes in Drosophila are generally not found adjacent to the Anopheles K+-
channel orthologs, suggesting that extensive translocation of genes has occurred since the
divergence of these two organisms.

The rapid rate of sequence acquisition has revolutionized
molecular biology. The sequencing of entire genomes, in
addition to new computer-based search tools has allowed us
to identify and analyze large sets of data very rapidly. The
acceleration of data acquisition, in fields such as whole-
genome sequence determination and genome-wide gene-
expression profiling, has opened the door for the study of
model organisms and organisms of importance to the study of

medicine and disease states by allowing for the analysis of the
entirety of genetic information in a given organism. The
recent completion of the sequencing of the Anopheles gam-
biae genome provides us with the entire genetic makeup of
this organism. Furthermore, the completion of the
sequencing of both the Drosophila melanogaster [1] and
Anopheles gambiae [2] genomes provides the first opportu-
nity for genome-wide comparisons from two metazoans from
the same order (Diptera). This presents new opportunities to

Genome Biology 2003, 4:R58

R58.2 Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack

detect synteny groups and facilitates the comparison of splic-
ing patterns and orthologous sequences between these two

The first K+ channel gene identified was cloned from Dro-
sophila. The Shaker gene was isolated by positional cloning of
a gene for which a mutation causes a leg-shaking phenotype
in anesthetized flies [3,4]. This gene encodes a six-transmem-
brane protein (Figure 1) subunit which assembles as a
tetramer. This gene provided a molecular probe by which
other K+ channel genes could be isolated by hybridization,
and later, by computer-based homology search. This led to
the cloning of different K+ channel subunits and the discovery
of different K+ channel types [5]. Subsequent to the cloning of
Shaker, K+ channel genes from the Shab, Shaw and Shal fam-
ilies (later renamed Kv2, Kv3, and Kv4, respectively, for clar-
ity [6]) were identified in Drosophila. These sequences are
shown in the alignment in Figure 2a and a tree is shown in
Figure 3a. Later, other types of K+ channel subunits were
identified by hybridization, with the conserved pore region
generally used as a probe, or by positional cloning using neu-
rological mutants in Drosophila melanogaster and other
organisms. Among these channel types were KQT channels,
calcium-activated K+ channels, inward rectifier K+ channels,
and the two-pore K+ channels [7]. The sequencing of the Dro-
sophila genome provided evidence that the vast majority of
K+ channel genes in the fruit fly have been identified, since
certain domains within K+ channels, particularly the pore
region, are readily identifiable by homology.

Other K+ channel types possess the same conserved pore
domain sequence as the Kv and KQT channels. Among the
six-transmembrane channels, there are two additional fami-
lies. The Eag gene family consists of eag, erg (seizure) and
elk; one of each is present in the Drosophila genome [8]. The
other 6TM K+ channel gene family is the Slo family. These
genes encode Ca2+-activated K+ channels of large conduct-
ance, intermediate conductance and small conductance:
these are thought to be mediated by Slo, slack and SK chan-
nels, respectively. These K+ channels are shown in Figure 2b.

Another family of two-transmembrane K+ channels called
inward rectifier K+ channels exists as well. Although these
channels lack a voltage-sensor domain they play an important
role in controlling resting potential and K+ homeostasis.
Between the two transmembrane domains these channels
possess a pore sequence homologous to the pore domain
found in Kv, Eag, and Slo channel types. Three Kir genes have
been reported in Drosophila [9]. Two of these genes, Irk2 and
Irk3, are quite similar at 54% amino acid sequence identity
while a third member is roughly 27% identical to the other
two. Finally, although they will not be investigated in this
study, a group of four-transmembrane, two-pore K+ channels
exists. These tandem-pore channels may be involved in a
wide range of physiologic processes but are generally thought
to mediate leak conductances which influence resting

Figure I
Membrane topology of K-channel subunits. The membrane topology of
the Kv-superfamily (a) and inward rectifier (b) channel subunits are
illustrated. The letter P is shown at the conserved pore domain and the
cytoplasmic amino and carboxyl termini for both types of channel subunits
are shown.

membrane potential. All the K+ channel genes mentioned
here contribute to K+ channel conductance in excitable and/
or non-excitable cells. In the nervous systems of insects and
other metazoans K+ channels are known to play an important
role in perception, learning and locomotion. This paper will
investigate the genes encoding K+ channels of two distantly
related Diptera now that their entire genomes have been
made public.

The entire set of predicted protein sequences from A. gam-
biae was downloaded from the National Center for Biotech-
nology Information [lo]. A Perl script was written to search
for proteins containing the conserved GYGD (single-letter
amino-acid code) K+-channel pore/selectivity filter motif. To
reconcile the fact that computer-generated open reading
frame (ORF) predictions might be imperfect I also used
TBLASTN to screen for proteins containing this pore region
using the amino-acid sequence of pore regions from the
major K+-channel families from Drosophila. Although a
definitive sequence analysis of full-length proteins cannot be
accomplished until the cloning of cDNAs and ESTs is carried
out, the high degree of similarity between the genes from

Genome Biology 2003, 4:R58

(a) K(v) superfamily

S1 S2 S3

(b) Inward rectifier


Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack R58.3

(8) I IVI I
(a 11 LnpK 1Y IEZA NLS TIQEYEDQAIA -------
Hu n I M w!a TI-EQY-KLATG Y-
Hu. CNQ2 I YEKSSEG------
Anoph w4 EVRPGRAG -------
H r41 reARRSSR--------
Anoph. E2 L D N - - - -
Hun 2.1 l LDEF------
DroI 3 II- PSG -----KILGN GR
A1 iipNQ.2 NMR-'YIRN ----- T
ApyIav- 3.1 -7,PL ATRVGT


TM2m TM3m

f~h-k~ OT 0 L 4RF TN I eL D
HunQ1---- TL]WEP A SA
"~ ~ ~ ~ ~ mW --- W L ETPFC1 e N.---

HF 21 EQPCGER FPQ l nDD --h-a
H-,-1 GQSTDNPQL- --------
Ip-ysa .1 YTWRLEKKETEPHE I EL ---------

---------- -V TS G I L M DVR
Hu ----2 VFATS RL D
Huu---- VLATS R VP

Anoph. . v4.VVT[NVD

Ar. ---- 1A i A GI
Dros 2 ------ R'D
A* ------------- I "GED
Au v2 1 ----------- HLD

AT oh, 312 ------- --- QI
Fig 9 e 7 4Q
Lobster" 3 IL N IQA

E* J*. E* E A

T, p KC Q G - --T
An K G PD--- LA

Hu N --- R-- A

LI GT -- ---- IS
Arop I GT- I
Hu, 14.1 KT -- -- I
I r o-----F--AC
ATophrF2 QT F A
KuH 2 1 DT --- ---
KI N P -------
Anphv33 NP DN -S
AoE31 NP e e
Anphv32 NP ----- ITT
I3 NP .... IT A
Lobs NP ...3 IT e
Ar IM I P *
Hu,~l. hS e

Figure 2 (see the legend on the page after next)

Genome Biology 2003, 4:R58

R58.4 Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack

TM1 m






iBF IC pipCIffPP~




i Fl
V Fi

Figure 2 (continued from the previous page, see the legend on the next page)

Genome Biology 2003, 4:R58

Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack R58.5

(C) TM1
AnophlRK22 -- -- -- --
DroslRK2 am *LPE ISG0
AnophlRK23 4*L QT D
CEL E I -- - - T1
RatKIR51 I DPD ------

AnophlRK31 4 *
AnophlRK32 **5 I *Q*
DroslRK3 4 QQSI"R
AnophlRK21 E
AnophlRK22 ** *E
MouseKIR62 *4 H
HumanKIR34 5****S'E
RatKIR51 I I a
HumKIR12 S K
HumKIR71 I I
E a -m M

AophIRK32 L IDE am E

Dm E. I WRM*
DroslRK ----EDR

R* *
I *n.
M ... IR22 G KGDR --





T l'i

AnohIRI E **I E

I IsK I 6 E I NI P

RatIR5 *I I I I1C i ~ll

Figure 2
Multiple alignment of channel sequence from Anopheles gambiae and other organisms. (a) Voltage-gated K-channel sequences; (b) Slo and Eag family K'
channels; (c) inward rectifier K' channels. Alignments were generated with the ClustalX program, and highlighted with BOXSHADE. Sequences were
chosen to illustrate diversity of Drosophila and mammalian K-channel types, in addition to the Anopheles sequences. Transmembrane domains are labeled
with a horizontal bar and the conserved GYG of the pore is marked by asterisks. The letter X represents regions at which amino-acid prediction was
particularly difficult in Anopheles sequences, generally because of short exons in coding regions. Anopheles sequences are predictions based on regions
homologous to Drosophila, as opposed to confirmed sequence data.

Genome Biology 2003, 4:R58

R58.6 Genome Biology 2003, Volume 4, Issue 9, Article R58

Figure 3
Phylogenetic trees of K+-channel types. (a) Voltage-dependent K+
channels; (b) inward rectifier K' channels. Sequences were aligned using
ClustalX. Six transmembrane channel sequences are confined to the
region spanning the first through the sixth transmembrane domain, so as
to remove highly variable sequence. Similarly, variable amino-terminal
sequences of inward rectifier K+ channels were removed in order to
exclude highly variable sequence. The culled alignments were then used to
construct a maximum likelihood tree in (a) and a neighbor-joining tree in
(b). The tree in (a) uses the Escherichia coli KCH K+ channel homolog
(Genbank accession number 808903) as an outgroup. For the tree in (b),
the bootstrap values above the branch before each node are based on
1,000 replicates and are a measure of robustness at each node. The Jones-
Taylor-Thornton amino-acid substitution matrix was used in the maximum
likelihood calculations from PHYLIP. The sequences in (b) are shown
without the punctuation such that MouseKIR22 = Mouse KIR2.2, and so
on. The sequence named CEL is Caenorhabditis elegans inward rectifier K+
channel (gi 7511460).

Anopheles and the well-characterized K+-channel genes in
Drosophila allows us to compare the genomic organization
and to predict coding regions with a high degree of

A series of BLAST searches [11,12] was carried out using the
amino-acid sequence at the Drosophila melanogaster
Shaker, Shab, Shaw, and Shal (Kvi, Kv2, Kv3, and Kv4) K-
channel pore region (from SwissProt Po8510, P17970,
P17971, P17972) as the query sequence against the DNA of the
Anopheles genome. In addition, a probabilistic ancestral
sequence (the most recent ancestor of the four major K-
channel families) was used as a query sequence with the hope
that more divergent sequences (for example, specialized K+-
channel types) might be identified. The first search, using the
amino acids spanning the Shaker K -channel pore sequence
from Drosophila as the query, revealed that the Shaker
ortholog in Anopheles is located at chromosome X:3D (see
Figure 4) and has 86% identity to the Drosophila gene prod-
uct (see Table 1). The Shaker gene in Drosophila is also
located on chromosome X, at 16F4. The Kvi gene in Anophe-
les had two 'pore' domains in close proximity on chromosome
X within genomic scaffold CRA x9PIGAV59NY 261. These
exons code for the amino acids 411-448 of the Drosophila
Shaker sequence. Closer scrutiny, and the observation that
other functionally critical segments of the coding region (such
as the voltage-sensor) were not redundant, led us to conclude
that these were splice variants, rather than separate genes.
This splice variant matched an exon already reported in the
spiny lobster Shaker K+ channel [13]. Another example of a
splice variant occurs at position 450-514 in Drosophila,
amino acids adjacent to the aforementioned exons at the pore
region, though it was not possible to find more than one
homologous sequence at this locus of the Anopheles genome
(see Figure 5). The organization of the gene in terms of
intron-exon boundaries was highly conserved between the
two species, with exons spanning DNA coding for amino-acid
positions 103-159 (110-159 in AG), 191-227, 257-297, 297-
348, 411-448, 450-513 observed in both species.

The Drosophila Shab sequence (SwissProt P17970) was used
as a query against the Anopheles genome. The Drosophila
gene Shab is located at chromosome 3L:63A1. The Anopheles
Shab (Kv2) ortholog lies at chromosome 2L:23C (see Table
2). The exon encoding the ORF spanning residues 256 to 438
of the Shab protein is conserved in Anopheles, though finding
an Anopheles sequence homologous to the amino-terminal
250 amino acids was not accomplished, perhaps because the
sequence is repetitive, particularly with respect to poly-
glutamine stretches, and, perhaps, species specific. These
homologs were found in Drosophila scaffold
142000013386045 section 10 (DNA sequence spanning
166595-194840) and Anopheles scaffold
CRA x9PIGAV5CJS_391 and _392. At least two other exons,
spanning residues 436-717 and 931-968, were also conserved
in both species. To look for microsynteny, CG9970, CG9972
and CG2077, putative genes products found directly
upstream and downstream of Shab in Drosophila, were run
against the Anopheles genome as queries in a TBLASTN
search. No homologous sequences in the mosquito genome

Genome Biology 2003, 4:R58


Irk 1,2 (4)

Irk3 (2)
Shaw (3)

elk eag SK

Shab KQT2

KQT1 slo

erg, C C
2L slack 2R 3L 3R X

Figure 4
Chromosomal locations of K-channel genes of Anopheles gambiae.
Triangles are colored red for highest sequence identity, green for
intermediate sequence identity, blue for lower sequence identity, and
purple for the inward rectifier genes. The 'hits' are based on sequence
similarity, with the Drosophila Shaw protein used as the query sequence
against the A. gambiae genome. The rectangular box indicates the location
of the highest score, reserved for the Anopheles ortholog of the query
sequence. C indicates the location of the centromeres.

were found at the same locus as Shab (Anopheles chromo-
some 2L:23).

I used the Drosophila Shaw (SwissProt P17972) sequence as
a query for homologs in Anopheles. The Shaw gene in Dro-
sophila is located at chromosome 2L:24A3-4. Not one but
three genes encoding K+-channel subunits of the Kv3 family
were present in the genome of Anopheles; these genes, ori-
ented in the same direction, were clustered at chromosome
3R:29, near the telomere (Figure 4), within a genomic
segment of roughly 150,ooo bases. The first was located at
region CRA x9P1GAV5CRW 227 and showed 85% amino-
acid identity, scaffold CRA x9P1GAV5CRW 225 showed
approximately 85% identity, and a third gene located at
CRA x9P1GAV5CRW 222 showed roughly 8o% identity. I
called these genes Kv3.1, Kv3.2, and Kv3.3, respectively. Sim-
ilar regions of protein sequence from a TBLASTN suggested
that the genes, particularly Kv3.1 and Kv3.2, have intron-
exon boundaries similar to those of the Drosophila Shaw
gene. As the sequence identity comparisons of Ag Kv3.1 vs
Dm Kv3.1 andAg Kv3.2 vs Dm Kv3.1 are nearly the same, the
assignment of the Anopheles 'ortholog' of Dm Kv3.1 is not
trivial: the ancestral sequence at the node which represents
the divergence of these two Anopheles genes is the actual
ortholog of Dm Kv3.1. The recent divergence ofAg Kv3.1 and
Ag Kv3.2 is supported by neighbor-joining, parsimony and
maximum-likelihood trees. The exons spanning amino-acid

Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack R58.7

positions 1-70, 109-175, 175-248, and 249-447 are present in
the Drosophila Shaw gene, as well as the two most similar
Anopheles genes, Kv3.1 and Kv3.2. The Anopheles Kv3.3 gene
has similar intron-exon boundaries compared to the Dro-
sophila K -channel ortholog. Exon-coding regions for amino-
acid positions 25-72, 72-116, 117-176, and 254-322 are
present in both the fly and mosquito, though other exons are
more variable between the two organisms. To look for
microsynteny in this region, I used Drosophila gene products
CG3513, CGlool9, CGo1002o, and cutlet, which flank the
Shaw locus in Drosophila, as queries to search for homologs
in the Anopheles genome. No homologous sequences mapped
to Anopheles chromosome 3R:29A.

I ran a BLAST search using the Anopheles Kv3.3 as the query
against the Drosophila genome, and this revealed a K+-chan-
nel sequence belonging to the Kv3 (Shaw) family located on
chromosome 2L:3oA8. This gene encodes a protein with 69%
amino-acid identity to the previously reported Shaw K+-chan-
nel sequence and 91% identity to the predicted Anopheles
Kv3.3 protein sequence, with a large percentage of amino-
acid differences confined to the short loop between the first
and second transmembrane domain, compared to the latter
sequence. For the purposes of this paper I will refer to the pre-
viously published Shaw sequence (SwissProt P17972) as

Table I

K* channel gene number and amino-acid sequence identity of
orthologs in Drosophila and Anopheles

Channel protein Drosophila Anopheles Amino-acid
identity (%)

Kvl I I 86
Kv2 I I 94
Kv3 2 3 87
Kv4 I I 96
KQT I 2 75

Eag I I 98
Erg I I 90
Elk I I 92
SIo I I 96
Slack I I 91
SK I I 94

IrkI I I 78
Irk2 I 3 65
Irk3 I 2 43

The middle two columns show the numbers of each gene for a given
K+-channel family in fruit fly and mosquito. The right-hand column gives
the percent amino-acid identity of the predicted gene product for each
gene in Drosophila against its ortholog in Anopheles. In cases for which
gene expansion in Anopheles made necessary two identity comparisons
(for example, Dm Kv3.I vs Ag Kv3.I and Ag Kv3.2), an average value
was used.

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R58.8 Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack


Figure 5
Conserved exon boundary for the pore domain of Kv I (Shaker) K-
channel genes. The splice variants are shown for Dros (Drosophila
melanogaster), Anoph (Anopheles gambiae) and Lobst, the spiny lobster,
Panulirus interruptus. Variations in sequence are boxed for emphasis.

Kv3.1 and the one I report here as Kv3.2. Dm Kv3.2 and Ag
Kv3.3 appear to define a new subfamily within the Kv3 K+-
channel family. The FlyBase GadFly Genome Annotation
Database [14] predicts that Kv3.2 (CG54450) spans 8,ooo
nucleotides and comprises at least 10 exons.

The Shal K+-channel sequence (SwissProt P17971) from Dro-
sophila was next used as a query sequence against both the
Anopheles and Drosophila genomes. The Shal gene in Dro-
sophila is located at chromosome 3L:76B. The Shal ortholog
was found at a region on chromosome 2L near 26C (Figure 4)
from Anopheles. Shal is found at Drosophila genomic scaf-
fold 142000013386050 section 52 and Anopheles scaffold
CRA x9P1GAV591D_309. There is considerable conserva-
tion of intron-exon boundaries between Drosophila and
Anopheles for these orthologs. An exon encoding amino acids
1-372 is present in Anopheles, but this region is split into two
exons in Drosophila from amino-acid position 1 to position
68 and another spanning amino-acid position 68 to position
372. Another exon spanning amino acids 440-488 is located
over 10 kilobases (kb) downstream in Anopheles, although in
Drosophila the corresponding exon is found approximately 1
kb downstream. An exon spanning the coding region for
amino acids 491-540 was found for both species. Evidence of
microsynteny was evident for the Shal locus between Dro-
sophila (chromosome 3L:76B5) andAnopheles (chromosome
2L:26). Gene products CG9231, CG9299, CG93oo and
CG9268, which lie in close proximity to Shal between
3L:76B3 and B5, showed regions of homologous sequence on
Anopheles chromosome 2L:26.

A highly conserved carboxy-terminal segment of KvLQT was
used as a query sequence for genes encoding K+ channels of
the KQT family. The carboxy-terminal sequence of mouse
KQT2 and the Caenorhabditis elegans KQT channel
(gi7511689) were run against the Anopheles genome. Homol-
ogous sequences were detected on Anopheles chromosome
3L:41A (scaffold AAABoolo8816 186) and twice on chromo-
some 2L:25 on adjacent scaffolds AAABooloo896o_650 and
651. Regarding the chromosome 2L homologous sequences,
the proximity of the two carboxy-terminal sequences and lack
of redundant sequence at other regions of this predicted pro-
tein suggest that these are splice variants rather than separate
genes. These same queries were run against the Drosophila

genome and just one homolog was found at genomic scaffold
142000013386047 section 13. This Drosophila gene product
KCNQ is roughly 75% identical to the Anopheles chromosome
2L gene product and 50% identical to the chromosome 3L
gene product, suggesting that the gene on chromosome 2L is
the Anopheles ortholog of Drosophila KCNQ. I will refer to
the chromosome 3 homolog as KCNQ2 (or KQT2) and the
chromosome 2 homolog as KCNQ1 (or KQT1).

Drosophila Slowpoke (gil7738179, chromosomal location
3R:96A) was used as a query against the Anopheles genome
using TBLASTN. There is a highly similar region at chromo-
some 2R:16A which is roughly 90% identical at the amino-
acid level on Anopheles genome scaffolds
AAABoloo8888_131 and AAABoloo8888_132. The exons
were short for these genes (typically encoding 12-50 amino
acids maximum) in Anopheles compared to what was
observed for the Kv channel genes. Slack and SK from Dro-
sophila revealed orthologs at chromosomal positions 2L:28D
and 3L:38C, respectively. I assembled the predicted
sequences from the exons. The Anopheles Slo, Slack, and SK
amino-acid sequences were 96, 91, and 94% identical,
respectively, to the Drosophila orthologs between the first
and sixth transmembrane domains.

Table 2

Chromosomal location and interarm homology of K+ channel

Drosophila Anopheles*

Kvl Shaker X:16F X:3D
Kv2 Shab 3L:63A 2L:23
Kv3 Shaw 2L:24C 3R:29 (x3)
2L:30A8 3R:29 (x3)
Kv4 Shal 3L:76B 2L:26
KQT I 2R:46F 2L:25
KQT 2 3L:41
Slowpoke 3R:96 A 2R:16
Slack 2R:47 A 2L:28D
SK X:4F 3L:38C
Eag X:13A 2R:13E
Erg (seizure) 2R:60B5 2L:28D
Elk 2R:55AI 2L:21F
Hyperkinetic X:9B 3R:37
IrkI 3R:94E 2R:7A
Irk2 3R:95A 2R:7A (x3)
Irk3 2L:37A 3R:29A (x2)

The locations of the different K+-channel genes are shown for
Anopheles gambiae and Drosophila melanogaster. Bold type is used for
those cases in which inter arm homology is conserved for the
orthologs. *The Anopheles chromosomal maps are still in their early
stages of annotation, making designations more general (estimated)
than for Drosophila.

Genome Biology 2003, 4:R58

A sequence nearly identical at the amino-acid level to Dro-
sophila Eag (gi17530941, chromosomal location X:13A) was
found at chromosome 2R:13E on scaffold
AAABoloo8859_213. The Drosophila Erg and Elk protein
sequences were used as queries and revealed orthologs in
Anopheles at chromosomal locations 2L:28D and 2L:21F,
respectively. The predicted Eag, Erg and Elk amino-acid
sequences from Drosophila were 98, 90, and 92% identical to
the Drosophila orthologs, respectively. Like the three genes
mentioned in the preceding paragraph, Eag, Elk and Erg were
encoded by exons much shorter than those observed for the
Kv K+-channel genes. The alignment for Slo- and Eag-family
K+ channels is shown in Figure 2b.

Each of the three Irk inward-rectifier K+-channel sequences
was used as a query against the Anopheles DNA database.
Four homologous genes were clustered very close together
near the telomere of chromosomal arm 2R at 2R:7A. Three of
these genes encode protein sequences very similar (nearly
70% amino-acid identity) to Drosophila Irk2. The fourth
gene, oriented in the opposite direction, was most similar to
Drosophila Dir (or Irk) (Figure 2c). Two additional genes
were located near the telomere of chromosomal arm 3R at
29A. These two genes, which have in common a large exon
encoding an ORF homologous to amino acids 144-437 of Dro-
sophila Irk3, were clustered very close to one another on the
chromosome; Irk3.1 and Irk3.2 from Anopheles, as I have
named these genes, are separated by no more than 1 kb of
intronic sequence. The predicted sequences share roughly
40% amino-acid identity to each of the three Drosophila
inward rectifier channels. It was necessary to consider
whether these two ORFs might constitute one two-pore chan-
nel but reciprocal BLAST searches using the Anopheles
sequences suggest that these sequences are most similar to
inward rectifier genes from Drosophila, and the presence of
carboxy-terminal signature sequences such as EILWGHRF
suggest the genes encode inward rectifier channels. The anal-
ysis of ORFs flanking Irk genes in the Drosophila genome
revealed that they were not in close proximity to the Irk
orthologs in Anopheles, again providing evidence of reshuf-
fling of genes in these two organisms.

A BLAST search using Drosophila Hyperkinetic (gi 902000,
chromosomal location X:9B) as the query revealed the pres-
ence of a K+ channel p-subunit ortholog in Anopheles
gambiae. This sequence was located at chromosome 3R:37D,
on scaffold AAABoloo898o_497. This sequence showed
roughly 78% amino-acid identity to the Drosophila ortholog
and 40-50% identity to the mammalian homologs. The amino
terminus of the Drosophila sequence is similar to the Anoph-
eles sequence of amino acids 162-200, upstream of the con-
served aldo-keto reductase core. Furthermore, the Anopheles
sequence, like Drosophila, has a histidine residue at the
position at which mammalian Shaker p-subunits have the
putative catalytic tyrosine.

Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack R58.9

Neutral evolutionary distance values
In addition to amino-acid identity we looked at neutral evolu-
tionary distance (NED) values. Values for f2 (the percentage
of identical codons for conserved twofold-redundant amino
acids Cys, Asp, Glu, Phe, His, Lys, Asn, Gln, and Tyr)
between two aligned proteins are calculated by looking at the
codons' third position in positions at which amino acids with
twofold degeneracy occur. These values may be more useful
for evaluating divergence dates than amino-acid sequence
identity because they are silent and mutation occurs in a
clocklike fashion, rather than in the bursts that are thought to
accompany rapid environmental changes. The f2 values for
Kvi, Kv2 and Kv4 orthologs in the fruit fly and mosquito were
as follows: 0.69 for Anopheles Kvi vs Drosophila Kvl, 0.73 for
Anopheles Kv2 vs Drosophila Kv2, and 0.69 for Anopheles
Kv4 vs Drosophila Kv4. The f2 values for Shaw vs Shaw and
other K+-channel genes were calculated, as shown in Table 3.
Anopheles Kv3.1 vs Anopheles Kv3.2 gave a f2 value of 0.74.
Anopheles Kv3.1 vs Anopheles Kv3.3 gave anf2 value of 0.75,
whereas Anopheles. Kv3.2 vs Anopheles Kv3.3 gave a value of
0.69. The f2 value of Anopheles Kv3.3 vs Drosophila Kv3.2
gave a value of only 0.52. The f2 value for the two Shaw genes
in Drosophila, Kv3.1 and Kv3.2, was o.6o.

Anopheles gambiae is the most important vector of Plasmo-
diumfalciparum malaria in Africa, where nearly 90% of the
world's malaria-specific mortality occurs. DDT has been used
extensively to control this mosquito. Because the target of
DDT and pyrethroid insecticides is the voltage-gated Na+
channel [15], and considering that the anti-malarial quinine
blocks K+ channels, insights into the ion channels in the
genomes of this mosquito and other insects may be useful for
investigating how DDT and other pesticides may be used with
greatest efficacy and safety. Using the conserved K+-channel
pore as a probe, I screened the entire A. gambiae genome for
the presence of voltage-gated K+ channels, Ca2 -activated K+
channels and inward rectifier K+ channels, as all these chan-
nels possess a homologous pore domain. I have identified
eight voltage-gated K+ channels, three Eag-family, three Slo-
family and six inward rectifier channel genes using this
search. A greater number of genes within a given family in
Anopheles compared to Drosophila can be a result of gene
expansion in Anopheles or, alternatively, gene loss in Dro-
sophila. I considered the likelihood of either possibility for
these cases, based on the trees that were constructed using
neighbor-joining, parsimony, and maximum-likelihood

K+ channels are dispersed throughout the genomes of both
Drosophila and Anopheles, although multiple members of a
given family are most often clustered. Comparing the gross
homology of the two species, both the Anopheles and Dro-
sophila have two major metacentric autosomes and an X
chromosome (five chromosomal arms in total). Of the

Genome Biology 2003, 4:R58

R58.10 Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack

Table 3

Third position (f2) values for the Kv3 K channels

Spec I Spec 2 f2 c2 n2

Mouse3.1 Aptero3.3 0.71 74 105
Hum3.1 0.85 116 136
LobstKv3 0.51 34 67
Dros3.1 0.58 40 69
Agam3.1 0.69 47 68
Agam3.2 0.75 47 63
Dros3.2 0.58 32 55
Agam3.3 0.66 41 62
Hum3.1 LobstKv3 0.54 65 121
Dros3.1 0.60 71 119
Agam3.1 0.52 60 115
Agam3.2 0.56 49 88
Dros3.2 0.54 53 99
Agam3.3 0.68 42 62
LobstKv3 Dros3.1 0.54 65 121
Agam3.1 0.60 71 119
Agam3.2 0.52 60 115
Dros3.2 0.56 49 88
Agam3.3 0.54 53 99
Dros3.1 Agam3.1 0.61 79 129
Agam3.2 0.65 81 124
Dros3.2 0.61 53 89
Agam3.3 0.62 62 100
Agam3.1 Agam3.2 0.74 93 126
Dros3.2 0.55 47 85
Agam3.3 0.75 72 96
Agam3.2 Dros3.2 0.57 47 83
Agam3.3 0.69 63 92
Dros3.2 Agam3.3 0.52 52 101
Dros Kvl AgamKvl 0.69 69 100
Dros Kv2 Agam Kv2 0.73 73 101
Dros Kv4 Agam Kv4 0.69 80 114

Calculations are based on alignments spanning the first through the
sixth transmembrane domain. The n2 value indicates number of
twofold degenerate amino acids, c2 indicates the number of twofold
degenerate amino acids with conserved third-position nucleotides, and
f2 signifies the percentage of conserved third-position nucleotides at
these positions (that is, c2/n2). Interspecies orthologs between fly and
mosquito (and between mouse and human, near top) are shown in
bold, and intraspecies paralogs are shown in underline and italics.
Comparisons of lower f2 values (for example, 0.55 vs 0.50) are not as
meaningful (with respect to estimating divergence dates) as higher-
value comparisons owing to equilibration within the lower ranges.
Agam, A. gambiae; Dros, D. melanogaster; Lobst, Panulirus interruptus;
Hum, Homo sapiens.

channels focused on here, only Shaker is located on the same
arm in both species, namely the X chromosome; however, the

locations of other K+ channel genes in Anopheles and Dro-
sophila are consistent with previously reported regions of
major interarm homology between these species (Table 2).
This was true for Shab (Dm 3L, Ag 2L), Shaw (Dm 2L, Ag
3R), Shal (Dm 3L, Ag 2L), KCNQ (Dm 2R, Ag 2L) and Slow-
poke (Dm 3R, Ag 2R), as well as for Slack, eag, erg, elk, and
the three inward rectifier genes, as shown in Table 2. The
translocations between autosomes and chromosome X,
observed for eag and Hyperkinetic, are notable: these exam-
ples raise questions about dosage compensation which will
need to be addressed in future studies.

There is 78-98% amino-acid sequence identity between the
six-transmembrane K -channel gene products in Drosophila
and their orthologs inAnopheles, a value significantly greater
than what other studies have calculated (62% identity and
56% in separate studies [16,17]) as a mean value for sequence
identity between orthologs in these two organisms. Amino-
acid sequence identity of 78-98% is an impressive figure,
given that these two organisms are thought to have diverged
250 million years ago [16]. Although this value may be slightly
higher than the true value, as uncertainties resulting from
splicing boundaries led us to disregard the more variable
amino-and carboxy-terminal extreme ends, the sequence
identities for the Drosophila and Anopheles K+-channel
orthologs over the core regions for K -channel sequences are
well above the mean values calculated by the other groups for
orthologs between these species. It suggests that K -channel
genes are subject to a stricter selection pressure than other
genes in these organisms. This is consistent with the observa-
tion that transporters and channels are among the proteins
with highest sequence similarity between Anopheles and
Drosophila [17].

Of the four voltage-gated K -channel types Kvl-4, the Shaker,
or Kvi channel gene is, from a genomic perspective, arguably
the most complex. Shaker from Drosophila is a gene with at
least 11 exons and spanning over 16 kb. Exons are short in the
Anopheles ortholog of Shaker as well, as it was not possible to
find an exon encoding more than 75 amino acids in this gene.
The presence of more than one pore region in Anopheles
Shaker suggests that sequence diversity can be generated in
an integral part of the internal segments of the channel,
rather than what has been reported for Drosophila Shaker -
splicing at the 5' and 3' ends [18,19]. Alternative splicing at
the pore region occurs in another arthropod, the lobster Pan-
ulirus interruptus. Functional channels translated from
genes with either of the two splice variants were expressed
and exhibited different electrophysiological and pharmaco-
logical properties [13]. It is tempting to assume that the two
transcripts with the two variable pore-regions in Anopheles
would encode channels with different properties as well,
although this would be premature until it is shown that both
exons are transcribed. An exon encoding the region
containing the pore exists in Drosophila, yet no transcripts
could be found containing this putative exon [20].

Genome Biology 2003, 4:R58

In the coding region, the Shal and Shab genes from Anophe-
les and Drosophila are made up of longer exons than the
Shaker gene. The lack of more than one splice variant at cen-
tral regions of the Shal protein suggests that splicing may be
confined to the 5' and 3' regions of this gene. Although evi-
dence of microsynteny was found for the region surrounding
Shal, flanking genes of the other channels did not provide evi-
dence of microsynteny between Anopheles and Drosophila at
these regions.

The identification of three Kv3 (Shaw) family K+-channel
genes inAnopheles (but only two in Drosophila) is intriguing.
In mammals, this family of K+ channels activates at potentials
considerably more positive than observed in other K+ channel
types; these channels have the ability to produce currents that
can specifically enable fast repolarization of action potentials
without compromising spike initiation or height [21]. Also,
these channels are localized at specialized regions in mamma-
lian brain associated with higher-order cognitive functions,
such as the thalamus and cortex [22]. Furthermore, Kv3
channel sequence identities are lower between Drosophila
and mammals than are other K+-channel types.

The identification of multiple Kv3 channel genes, but only
single members of the Kvi, Kv2 and Kv4 families, in Diptera
(two in Drosophila and three in Anopheles) raises questions
about the evolutionary history of Kv3 K+ channel genes. In
some organisms with very primitive nervous systems, such as
Polyorchis penicillatus (jellyfish, phylum Cnidaria) at least
two Shaker (Kvl)-family genes exist [23]; moreover, in the
electric fish Apteronotus the Kvi (Shaker) family is the most
diverse, with at least 10 members [24]. One can predict from
the protein and DNA similarity that Kv3.1 and Kv3.2 from
Anopheles diverged recently. The previously published Dro-
sophila Kv3 (Shaw) protein is 88% identical to the Anopheles
Kv3.1 and Kv3.2 sequences, but only 70% identical to the
Anopheles predicted Kv3.3 gene product. Regarding the rela-
tionships between Dm Kv3.1 and the two Anopheles genes Ag
Kv3.1 and Ag Kv3.2, this paper has already stated that the
ancestral sequence at the node representing the divergence of
these two Anopheles genes is the true ortholog of Dm Kv3.1
(gi 158460). Given the awkwardness of comparing an extant
gene (for example Dm Kv3.1) to its ancestral ortholog, it may
suit the genomics and/or evolutionary community to devise
new terminology for such cases. In relation to the Drosophila
Kv3.1, the terms 'novolog' (corresponding to Ag Kv3.1 or Ag
Kv3.2) and 'archaelog' (corresponding to the ancestral gene
represented by the node from which the two Anopheles genes
diverged) might be useful; these terms, as presented here,
would apply to cases in which contemporaneous orthologs do
not exist between two organisms, as opposed to the general
phenomena of duplication and divergence.

In the light of the high amino-acid identity, roughly 87%, the
low f2 value of 0.51 for Anopheles Kv3.3 and its ortholog in
Drosophila (as opposed to an f2 value of 0.69 for Anopheles

Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack R58.1 I

Kvi vs Drosophila Kvi, 0.73 for Anopheles Kv2 vs Drosophila
Kv2, or 0.70 for Anopheles Kv4 vs Drosophila Kv4) suggests
these two genes diverged longer ago than would be predicted
by the amino-acid identity alone, and that selective pressure
has prevented the two sequences from diverging; homoplasy
may explain their high amino-acid identity and low third-
position (f2) identity. The f2 value comparing Kv3.1 vs Kv3.3
from the mosquito is 0.75, higher than expected, considering
that amino-acid identity between the two (64%) is
significantly lower than that observed between Anopheles
Kv3.3 and its Drosophila ortholog.

It is likely that different K+ channel subunits within the same
family would provide the potential to generate many K+-
channel tetramer combinations. This would allow greater
variation and specificity of Kv3 channels, as K+ channel subu-
nits within a family can readily form functional heteromul-
timeric channels [25]. The number of XXR repeats of the
voltage-sensor (where X is a hydrophobic residue and R rep-
resents arginine within the voltage-sensor) in non-vertebrate
Kv3 K+ channels is of interest. The presence of four such
repeats in invertebrate Kv3 channels and six in vertebrate
Kv3 K+ channels may help explain the difference in voltage-
dependence observed between the mammalian and fly Kv3
channels, as even single amino-acid mutations in this domain
can affect voltage-dependence of K+ channels considerably
[26]. The greater PAM distance between Drosophila and
mammalian Kv3 channels (PAM distance 65) compared to
Kvi, Kv2, or Kv4 (for which the intra-family PAM distances
between Drosophila and mammalian channel sequences
range from 25-40) shows that Kv3 channels have undergone
more extensive adaptation than other K+-channel families. It
can be inferred that the greater complexity of the vertebrate
brain made necessary a rapidly deactivating, high-threshold
K+-channel type which has not evolved in protostomes;
indeed, given the biophysical properties of Kv3 channels in
mammals, the amino-acid replacements that have occurred
in mammalian Kv3 channels seem to have provided exactly

Like the Kv3 (Shaw) family, KQT (KCNQ) K+-channel genes
are more abundant in Anopheles than in Drosophila.
Sequence analysis suggests these channels evolved before
other classical voltage-gated K+ channels (Kvl-K4). The
neighbor-joining (Figure 3a) and maximum-likelihood trees
we constructed, in combination with the fact that mammalian
KCNQ1 and Anopheles KCNQ2 gene products share a striking
75% identity (despite the divergence of protostomes and deu-
terostomes close to 700 million years ago), suggest that gene
loss in Drosophila, specifically loss of an ancestral KCNQ2
(mammalian KCNQ1), is the cause of this difference, rather
than gene expansion in Anopheles, which may be the case for
the Kv3 and Irk3 (Figure 3b) gene families. Alternatively,
lateral transfer of KCNQ1 from mammals to Anopheles must
be considered, given the intimate relationship of these organ-
isms. Although the genome size of Anopheles is twice the size

Genome Biology 2003, 4:R58

R58.12 Genome Biology 2003, Volume 4, Issue 9, Article R58 McCormack

of the Drosophila genome, the numbers of genes in both
organisms are nearly equivalent [17], suggesting that gene
duplication depends on the advantage of additional genes in
distinct families, rather than a general consequence of pos-
sessing a larger genome. Unlike other K+-channel types, for
which amino and carboxyl termini are highly variable, KQT
channel sequences are even more highly conserved in some
regions of the cytoplasmic carboxy-terminal region than in
the conserved pore region. The presence of two potential
splice variants within the carboxy-terminal tail raises ques-
tions about the role of this domain in channel function.
Although the physiologic significance of this region is not yet
known, evidence suggests it may be involved in calmodulin
binding [27]. For this region one homologous gene product
can be found in Drosophila, KCNQ, which raises questions
about whether products of this gene mediate the M-current,
as has been postulated for KCNQ2 and KCNQ3 in mammals

The greater number of inward rectifier K-channel genes in
Anopheles compared to Drosophila is striking, given that
these organisms belong to the same order. Our analysis,
based on maximum-likelihood and neighbor-joining
algorithms, suggests that gene duplication inAnopheles is the
most likely explanation for the greater number of Irk3 genes
in mosquito. This also appears to be the case for Irk2.1 and
Irk2.2; however, from the tree (Figure 3b) it appears that the
divergence of Irk2.3 from Irk2.1 and Irk2.2 in the mosquito
occurred earlier than the divergence of Drosophila Irk2 and
the two Anopheles genes Irk2.1 and Irk2.1, suggesting that
gene loss in Drosophila may have occurred. The same tree
topology was supported by both neighbor-joining and maxi-
mum-likelihood trees, though lack of a clear ortholog from a
more distant organism (for example, a deuterostome) makes
this type of assessment, regarding gene history, more diffi-
cult. Future studies may help explain why the mosquito has
twice as many of the inward rectifier genes as the fruit fly.
Gene expansion in Anopheles has been observed for genes
involved in hematophagy and insecticide resistance; it is
unclear to what extent these two factors are involved here,
although ion channels are clearly targets of insecticides. The
overall compositions of K-channel genes in Anopheles and
Drosophila are strikingly similar in some respects, such as
conservation of sequence and intron-exon boundaries of
orthologs, and strikingly different in others, such as the
number of Irk homologs and lack of microsynteny. The
genome projects of other insects, such as Manduca sexta and
Bombyx mori, will help paint a broader picture of the compo-
sition of ion-channel genes within the genomes of these
related organisms.

Within the Anopheles genome there are orthologs for the four
major voltage-dependent K+-channel gene families in Dro-
sophila: Kvi, Kv2, Kv3 and Kv4 (Shaker, Shab, Shaw and

Shal, respectively). In addition we have identified genes that
encode the Shaker p-subunit, two members of the KQT family
of K+ channels, as well as three Slo-family genes, three Eag-
family genes, and six inward rectifier K+-channel genes. In
Anopheles, the Shaw family is more diverse than in Dro-
sophila: three genes from this family are located next to one
another along chromosome 3R, in contrast to two Kv3-family
genes in Drosophila. The greater number of genes for three
K+-channel types, inward rectifier, KQT, and Kv3 (Shaw), in
Anopheles is intriguing, given that these organisms have
roughly the same number of genes: both gene expansion in
Anopheles and gene loss in Drosophila, in separate cases,
may account for these differences. The high level of amino-
acid sequence identity, as well as the conservation of intron-
exon boundaries, in combination with the chromosomal
proximity of these genes in Anopheles and Drosophila, pro-
vides a greater understanding of the molecular diversity and
evolutionary history of K+-channel genes in the order Diptera.

Materials and methods
I used BLAST [11] and PSI-BLAST at the NCBI website to find
K+-channel homologs using the Shaker K-channel pore
sequence as a query initially and then other, longer, K+-chan-
nel family-specific query sequences for verification. The pre-
dicted splice sites were compared with results of the
TBLASTN to help confirm intron-exon boundaries. Increases
in nucleotide position number from one putative exon to the
next were used to deduce the size of introns.

This study utilized the Ensembl Anopheles gambiae server
[29] to search for homologs of various K+-channel types and
to identify and visualize their respective chromosomal loca-
tions. The DARWIN server [30] was used to calculate the f2
values for the sequences, as well as a phylogenetic tree for the
Shaw sequences, along with PAM distances and ancestral
sequences. Figures were visualized and optimized using
Adobe Photoshop.

Sequences were aligned using ClustalX version 1.81. Phyloge-
netic trees were generated using ClustalX (for neighbor join-
ing) and PHYLIP (for neighbor joining, parsimony and
maximum likelihood using Protdist, Protpars, and ProML,
respectively). The resulting trees were then visualized and
evaluated using Treeview. Bootstrap values were calculated
using ClustalX and PHYLIP.

I thank Steven Benner, David Schreiber, Eric Gaucher and Ken McCormack
for their advice and critical evaluations of the manuscript. I thank Yando Del
Rio for advice and inspiration.

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