Download A component of calcium-activated potassium channels encoded by

A Component of Calcium-Activated Potassium Channels Encoded by the
Drosophila slo Locus
Nigel S. Atkinson; Gail A. Robertson; Barry Ganetzky
Science, New Series, Vol. 253, No. 5019. (Aug. 2, 1991), pp. 551-555.
Stable URL:
http://links.jstor.org/sici?sici=0036-8075%2819910802%293%3A253%3A5019%3C551%3AACOCPC%3E2.0.CO%3B2-D
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J. Bowman, C. M. Yentsch, W. T. Peterson, Eds.
(Springer-Verlag,Berlin, 1986), pp. 193-223.
19. The processes responsible for the shape of the curves
in Fig. 2 are not known.
20. P. F. Sale, Enoiron. Biol. Fish. 3, 85 (1978); P. K.
Dayton, in Erological Procwec in C o m l and Marine
System, R. J. Livingston, Ed. (Plenum Press, New
York 1979),pp. 3-18; M. A. Kendall, R. S. Bowman,
P. Williamson, J. R. Lewis, Neth. J. Sea Res. 16, 119
(1982); A. J. Underwood andE. J. Denley, inEcobgual
Communities: Conrep~ualIrnw and the Evidence, D. R.
Strong, Jr., D. Simberloff, L. G. Abele, A. B. Thistle,
Eds. (Princeton Univ. Press, Princeton, NJ, 1984), pp.
151-180; J. Roughgarden, S. Gaines, H. Possmgham,
ScMlre 241, 1460 (1988).
21. P. M. Yoshioka, Ecology 63, 457 (1982).
22. Because of the obviously nonnormal distribution of
the data a nonparameaic test was used. Each daily
settlement value consisted of the average of two or
three settlement plates per site. One site was observed from 29 April until 8 August 1989; the
Spearman correlation coefficients between settlement and water temperature anomaly were -0.258
for Chthamalus and -0.223 for Polliripes (overall),
whereas those from 1 June to 29 August were
-0.266 and -0.400. Coefficients from the other
site, observed from 3 June until 27 July, were
23.
24.
25.
26.
27.
-0.274 for Chthamalw and -0.482 for IJollicipes. All
correlation c d c i e n ~
are signilicantly different from 0
at a = 0.025 (one tail). Small magnitude of correlation
values might reflect uncertainty of the offshore larval
reservoir, differential behavior of internal bores at the
Scripps Pier and Dyke R& unrecorded large-event
bores, or other phenomena affecting settlement.
L. R. Haury, M. G. Briscoe, M. H. Orr, Nature 278,
312 (1979).
A. M. Barnett and A. E. Jahn, Cont. SheFRes. 7, 1
(1987).
P. E. Smith and R. W. Eppley, Limnol. Oceanogr.
27, 1 (1982).
J. Pineda, unpublished observations.
I thank P. Dayton, J. Enright, L. Haury, M. Tegner, E.
Vetter, and C. Wlnant, and two reviewers for consauctive comments on the manuscript. I also thank many
divers who helped sample. Temperame data were
supplied by the ScrippsInstitutionof Omography. E.
del Castillo supported me in every stage of the research.
I especially wish to thank the people of Mexico who
supported this research through a CONACYTgraduate
fellowship which also covered research expenses. I also
arknowledge grants from NSF m P. K. Dayton and
from MMS to C. D. Winant.
4 February 1991; accepted 16 May 1991
A Component of Calcium-Activated Potassium
Channels Encoded by the Drosophila slo Locus
Calcium-activated potassium channels mediate many biologically important functions
in electrically excitable cells. Despite recent'progress in the molecular analysis of
voltage-activated K+ channels, Ca2+-activated K+ channels have not been similarly
characterized. The Drosophila slowpoke (slo) locus, mutations of which specitically
abolish a Ca2+-activated K+ current in muscles and neurons, provides an opportunity
for molecular characterization of these channels. Genornic and complementary DNA
clones &om the slo locus were'isolated and sequenced. The polypeptide predicted by slo
is similar to voltage-activated K+ channel polypeptidesin discrete domains known to
be essential for function. Thus, these results indicate that slo encodes a structural
component of Ca2+-activated K+ channels.
P
OTASSIUM CHANNELS COMPRISE A
large and diverse group of integral
membrane proteins that determine
the level of excitability and the repolarization properties of neurons and muscle fibers
(1). On the basis of gating mechanisms K+
channels can be subdivided into at least two
distinct classes: voltage-activated K+ channels respond to changes in membrane potential, whereas the gating of Ca2+-activated
KC channels is dependent on intracellular
Ca2+, although members of this class are
also sensitive to voltage. Ca2+-activatedK+
channels occur in neurons, muscles, and
secretory cells of vertebrates and invertebrates and govern action potential repolarization, the rate and pattern of repetitive
firing, and Ca2+ entry ( 2 ) .
Laboratory of Genetics, University of Wisconsin, Madison, WI 53706.
*Present address: Department of Zoology, University of
Texas, Austin, TX 78712.
2 AUGUST 1991
The molecular analysis of KC channels
was initiated by the cloning of the Drosophila Shaker ( ~ h locus,
)
a gene encoding
one class of voltage-activated KC channels
(3). Subsequently, many KC channel genes
were cloned from various organisms on the
basis of sequence similarity with Sh, enabling elucidation of important structural
features essential to K+ channel activity (4).
However, none of these genes appears to
encode a K+ channel gated by Ca2+
In Drosophila, a fast, Ca2+-activatedKC
current, I,,, has been identified in adult and
larval muscles and in 'larval neurons (5-7).
Like the BK class of Ca2+-activated K+
channels in mammalian muscle (8), the
Drosophila channels that conduct I,, are
sensitive to charybdotoxin and have a relatively large single channel conductance (6,
7). Mutations of the slo locus specifically
eliminate I,, (6, 7, 9). To determine if slo
encodes a structural component of Ca2+activated K+ channels or perhaps affects
their function by some other means, we
undertook a molecular analysis of this locus
(10).
We first pinpointed the cytological location of the "gene., defined on the basis of a
single, cytologically normal mutation, slol
(6). We mapped this mutation by meiotic
recombination to position 90.0 on the third
chromosome between bar-3 (map position
79.1) and taxi (map position 91), corresponding to polytene chromosome region
95-96 (11).To refine this location, we isolated three y-ray induced slo mutations (slo3,
slo4, and slog) on the basis of their failure to
complement the slol behavioral phenotype
(6, 12) (Table 1).The slo phenotype of these
new mutations was confirmed electrophysiologically (13).
&ological examination of these mutations revealed that all contained chromosome rearrangements with a common breakpoint at polytene band 96A17 (Table 1).
These results together with the cytological
and genetic characterization of other chromosome lesions in this region, originally
isolated by other criteria, identify band
96A17 as the cytological location of slo
(Table 1).
One of the breakpoints in the slo4 inversion is at the slo locus and the other is at
96F5-8 close to the previously cloned E(sp1)
locus (14). We were thus able to k e this
invirsibn'to clone genomic DNA from the
slo locus by chromosome walking and jumping (15). Chromosomal in situ hybridization
o f t w o sets of phage clones from existing
chromosome walks in the E(sp1) region revealed that one set was proximal and the
other set distal to the 96F5-8 breakpoint.
Consequently, we extended the proximal
walk by isolating overlapping cosmids from
a wild-type library until the 96F5-8 break~ o i n t&as reached. To identifv a cosmid
spanning the inversion breakpoint (Fig.
lA), we assayed each cosmid from the walk
>
,
Table 1. Summary of chromosome aberrations
used in cytological mapping of the slo locus.
- --
Name
-
Cytological lesion
Normal
Deletion (95E6-7
to 96A18)
do4
Inversion (96A17
and 96F5-8)
slo8
Deletion (96A2-9
to 96D2-4)
Df(3R)S87-5 Deletion (95F7-9
to 96A17-18)
Df(3R)S87-4 Deletion (95E8-F1
to 95F15-96A2)
T(Y;3)AI 17 Translocation (broken
at 96A10-17)
slo'
S ~ O ~
slo
phenotype
Mutant
Mutant
Mutant
Mutant
Mutant
Normal
Mutant
REPORTS
551
by chromosomal in situ hybridization to slo4
homozygotes: a cosmid spanning the breakpoint should hybridize to both ends of the
inversion, whereas cosmids that stop short
of this break should hybridize to a single
site. DNA disrupted by the inversion was
further delimited by in situ hybridization
and Southern blot analysis to a 3-kb Eco RI
restriction fiagrnent within this cosmid. In
slo4, the 3-kb Eco RI genomic fragment at
96F5-8 is broken into two parts, each juxtaposed to DNA sequences from the slo
locus at 96A17. A phage containing one of
these junction fiagments was isolated by
probing a slo4 genomic library with the 3-kb
wild-type Eco RI fragment. This junction
fragment was subsequently used to initiate a
chromosome walk in a wild-type cosmid
library to obtain genomic DNA that induded the slo locus (Fig. 1B).
An approximate location of the slo transcription unit was defined by the 96A breakpoint of slo4, which mapped to a 3-kb Eco
RI-Hind 111 genomic fiagment. We used
this fragment to screen cDNA libraries from
-
-10
I
I
p yl
$?
Y
1
1 1 1 I
1
1 1 1 1
I
I
-
53-'
1
I
2p
3p
-L
I
I
;;
;'I
Fig. 1. Cloning of genomic DNA from the slo
locus. (A) Hybridization of a cosmid from the
genomic wak to polytene chromosomes of slo4
homozygotes (34). (8) An EEO RI map of the
cloned region. The position of the slo4 inversion
breakpoint is indicated. H, the Hind I11 site that
marks the rightward limit of the resmction fragment containing the breakpoint; black bars, the
genomic resmction fragments that hybridize with
slo CDNAs 254, 28,and CH4 (see text and Fig.
3). (The presence of a black bar indicates only that
the corresponding resmction fragment above the
bar contains one or more exons of unknown size.
Because the CDNAs lack internal Eco RI sites, a
break berween bars indicates the presence of an
intron in genomic DNA that is spliced out in the
cDNAs.) Broken lines, regions where the order
of the resmction fragments has not been determined so the distance between exons is not
known. The entire slo locus must extend beyond
the limits shown because the cDNAs hybridize to
genomic resmction fragments not contained in
the cloned region and because the T(Y;3)A117
breakpoint lies outside the limits of the chromosome walk. The direction of transcription relative
to the chromosome wak was determined from
Northern blots by using single-stranded RNA
probes.
Drosophila heads. One DNA, 28, hybridizcd to both sides of the slo4 inversion and
also to both parts of the T(Y;3)A117translocation (Fig. 2, A and B). The fact that 28
spans two different slo mutations demonstrates that this cDNA is derived fiom the
slo transcript. Two transcripts of 5.8 kb and
11kb are detected by 28 probes on North-
ern blots of polyadenylated [poly(A)+]
RNA isolated fiom heads (Fig. 2C). The
absence of these transcripts in slo4 homozygotes (Fig. 2C) identifies them as slo transcripts. We do not know whether the two
wild-type transcripts encode different slo
polypeptides.
Because 28 is incomplete (see below), we
Fig. 2. Identification of the slo transcription unit.
IA) Hvbridization of d l N A 28 to wlvtene chro;ndsokes homozygous for the slo4*&ersion. Arrowheads, hybridization signals at inversion end
points. (8) Hybridization of CDNA 28 to polytene chromosomes heterozygous for the
T(Y;3)A117 translocation and a normal third
chromosome. A hvbridization s i d (arrowhead)
is seen at the &tact sfo locusYon ;he normal
chromosome. In addition, both the proximal
(double arrowhead) and distal parts ( 0 6 arrow)
of the translocated chromosome contain sites that
hybridize. In this preparation part of the distal
piece of the translocation has asynapsed from the
normal chromosome. (C) Northern blot analysis
of slo transcripts. Approximately 10 pg each of
poly(A)+ RNA isolated from heads of Canton S
(CS) and homozygous slo4 adults was loaded into
individual lanes. The filter was probed with 32Plabeled cDNA 28 (left). S i results were
obtained when blots' w&e probed with either
*m
.
I
I
I
purified fragments of 28 lacking any intron sequenccs or single-stranded RNA transcribed from the T7promoter of the pBluescript SK- (Stratagene)
plasmid that carried cDNA ZS.Single-stranded probes transcribed in the reverse orientation from the
SP6 promoter of 28 did not detect any nanscripts (not shown). As a control, the blot was stripped and
reprobed with an eag cDNA (right). Experimental conditions were as described (26).
Composite
CDNA
'
0
S1' S2S3 S4SSWS8
254
I
exonA exonB y - t h d i n g ?
I
Z8
I
I
V
I
500
Amlm .cldmldw
A
M
M
A
M
A
A
CH4
loo0
Fig. 3. Diagrammatic representation and hydrophobicity plot of overlapping slo cDNAs that were
assembled to yield a composite sequence. (A) The three CDNAs drawn to scale. Dashed underlines,
CDNA segments that were assembled into the composite shown below; open boxes, open reading
frames; hatched boxes, introns that were not spliced out of some CDNAs (17). The open reading frame
of 254 is preceded by an untranslated segment (not shown) that may also represent an unspliced intron
(17). ALI the regions shown have been completely sequenced on both strands (16), with the exception
of the central segment of 254 (interrupted bar), which has not been sequenced. For those regions that
have been fully sequenced, the nucleotide sequences in overlapping segments are identical except for the
unspliced i n m and the two sites of alternative splicing indicated. At site A an exon encoding 14
amino acids (Al) that is present in CH4 is replaced by a different exon encodmg 25 amino acids (A2)
in 28.At site B an exon 22 amino acids long is present in 254 and 28 but lacking in CH4.28, which
does not end in a poly(A)+ tail, extends beyond the poly(A)+ tail present in CH4 suggesting the
presence of alternative polyadenylationsites. (8) Hydropathy plot for the deducedslo polypeptide (Fig.
4) encoded by the composite cDNA. The plot has been aligned with the composite cDNA sequence
above. Hydrophobicity values were calculated accordmg to Kyte and Doolittle (19) with a window size
of nine amino acids. The seven shaded hydrophobic peaks were named according to the corresponding
domains of other K+ channels after aligning sequences of the fourth and sixth hydrophobic peaks with
S4 and H5, respectively.
SCIENCE, VOL. 253
hydrophobic domains near the NH,-terminus of the slo polypeptide (Slo) similar to
the structure of other known K+ channel
polypeptides (4). The fourth hydrophobic
segment of Slo is similar to the S4'domainin
voltage-gated channels, which is thought to
mediatevoltage sensitivity. The S4 m&f is
characterized by a positively charged residue
at every third position separated by two
hydrophobic residues (20). Out of 26 positions in the alignment of S4 domains from
Slo and Shaker, there are six amino acid
1 m G C D Q S T V ESLADDPTDS PFDADDCLKV RmWCFLLSS IFTFLAGLLV
Fig'
Deduced
acid se- identities and four conservative substituSI - quence encoded by a composite slo
51 VLLWRAFAFV CCRKEPDLGP NDPKQKEQKA SRNKQEFEGT FMTEAKDWAG
A
cDNA. The hydrophobic domains tions. This region of Slo shows a slightly
101 ELISGQTTTG RILVVLVFIL SIASLIIYFV DASSEEVERC QXWSNNITQP
are underlined. &sensus sites are better alignment with an S4 domain of rat
-aindicated for CAMP phosphoryla- brain Na+ channels, yielding seven identi151 IDLAFNIFFM VYFFIRFIAA SDKLWFMLEM YSFVDYFTIP PSFVSIYLDR
.
Q
I
tion (W) (35), PKC-dependent ties and four conservauvesubstitutions (Fig.
201 TWIGLRFLRA LNNTVPDIL QYLNVLKTSS SIRLAQLVSI FISVWLTAAG
phosphorylation (#) (35), a potenS
4
- S S
# '
251 IIRLLENSGD PLDFDNAHRL SYWTCVYFLI VTMSTVGYGD VYCETVLGRT
tial ATP-bindingsite (e)(31), and 5A). The Slo S4 domain contains fewer
I
B
'
L
a region containing a potential positive charges than S4 domains in voltage301 FLVFFLLVGL AMFASSIPEI IELVGSGNm GGELKREHGK RAIWCGRIT
s
6
Ca2+-bindingloop (*) (30). Below gated channels but the hnctional signifi351 YESVSAFLKD FLREDREDW VEVVFLRRKP PDLELEGLFK RAFTTVEFFQ
are the deduced amino acid se- cance of this is not known. The presence of
401 GTIMNPIDLQ RWNREADAC L W C Q D PDAEDRANIM RVISIKNYSD
quences of two alternative exons
#
not included in the composite se- an S4 domain in a guanosine 3',5'-mono451 DIRVIIQW YRNKAYLLNI PSWDWKQGDD VICLAELKLG FIAQSCLAPG
quence. Alternative exon A2 is phosphate (cGMP)-gated channel has led to
501 FSTMMANLFA MRSFKTSPDM QSWTNDYLRG TGMEMYTETL SPTFIGIPFA
found in cDNA 28 in the place of the suggestionthat the S4 sequence contrib#
551 QATELCFSKL KLLLLhIEIK GAEEGADSKI SINPRGAKIQ ANTWFFIAQ
exon Al. Alternative exon B is utes to the basic architecture of various
found in cDNAs 28 and 254 but channels in addition to its function as a
601 SADEVRRAWF Y C W H E D I K DETLIKKCKC KNLTVQPRSK FDDLDEAAPA
L N t k A l 2
missing in CH4. The single letter
651 PTFTPPELPK RVRVRGSVSG DITRDREDTN LLNRNVRReN GTGNGTGGMH
abbreviations for amino acids are voltage sensor (21) .
701 HbUWTAAAAA AAAAAGKQVN KVKPTVNVSR QVEGQVISPS QYNRPTSRSS
The greatest similarity between Slo and
A, Ala; C, Cys; D, Asp; E, Glu; F,
NLEmaB f #
751 GTGTQNQNGG VSLPAGIADD QSKDFDFEKT EMRlDSTGMF HWSPAKSLED
Phe; G, Gly; H , His; I, Ile; K, Lys; K+ channel polypeptides is in the H5 do** *
L, Leu; M, Met; N, Asn; P, Pro; Q, main, an integral part of the ion conduction
801 CILDRNQAAM T V L N G W C LFADPDSPLI GLRNLVMPLR ASNFRYRELK
Gln; R, Arg; S, Ser; T, Thr; V, Val;
851 H W I V G S W Y IRREWi+%QN LPKISVLNGS PLSRADLRAV NVNLCDMCCI
W, Trp; and Y, Tyr. The nucleotide pore (22-25). When aligned with an H 5
901 LSAKVPSNDD PTLADKEAIL A S L N I M F DDTIGVLSQR GPEFDNLSAT
sequence of the composite cDNA consensus sequence for the Sh superfamily,
#
#
has been submitted to GenBank Slo shares 11identities and five conservative
951 AGSPIVLQPR GSVYGANVPM ITELVNDGNV QFLDQDDDDD PDTELYLTQP
under accession number M69053.
substitutions out of 25 positions (Fig. 5B).
1001 FACGTAFAVS VLDSLMSTTY FNPNALTLIR SLITGGATPE LELILAEGAG
This alignment is centered on a core of 11
1051 LRGGYSTVES LSNRDRCRVG QISLYDGPLA QFGECGKYGD LFVAALKSYG
#
amino acids in which there are nine identi1101 MLCIGLYRFR DTSSSCDASS KRYVITNPPD DFSLLPTDQV FJTNQFDPGL
ties and two conservative substitutions.
##
1151 EYXPPAVRAP AGGRGTNTQG SGVGGGGSNK DDNS
Moreover, at five positions in the H 5 do#
main where Slo deviates from the consensus,
Alternative exon A2
it shares identities with Eag, another Dro632 ATFRKGVPAVQMVGRAKDDEYSLSN
sophila K+ channel polypeptide identified by
means of genetic strategies (26, 27). These
Alternative exon B
745 PPENDANPYAGYQLAYEW
relations indicate that slo and other K+
channel genes have evolved from a common
ancestor.
One of the conservative changes within
Fig. 5. Alignment of hydrophobic
.,
domains S4 and H5 encoded by slo
x c m
the H5 domain of Slowith respect to the Sh
with corresponding segments of
family consensus is a substitution of serine
D I L Q
other channel polypeptides. (A)
for threonine at wsition 284. Creation of
P G L K
Alignment of S4 domains of Sh K+
the same replacement in Sh by site-directed
channel (top) (6, 25), rat brain Na+
channel type I (R,) (bottom) (36),
mutagenesis results in a tenfold decrease in
and slo (middle).Amino acid idenB
sensitivity to internal tetraethylammonium
tities are shaded and conservative
sh
T
~ c V
F w 8
(TEA) blockade (23). This result is consissubstimtions are boxed. The posi- ~ K I. ,
". D A F ~ W A V V S Y T T V G Y tent
G Dwith
H the
Y Pobservation
VFIF
G in vivo, I,, is
that,
tive gating charges in the S4 doBE2 ID$
S-UW,,A V V, S M T T .V G Y G D H V P T T I G G! relatively insensitive to internal TEA
main of Sh and rat Na+ channel are
indicated. The charges in parenthe- RCKS D " A . F ~ R . W - A ? V , T H T T V G x G D H B P V T I G G
(K,= 100 rnM) (28). Site-directed muta,-I'
.
ses are those present in the Sh and RCIM
tions in Sh that alter ionic selectivity and
+% w ?:A v v:"' M
~ V GT- Y - G D M K P I T v G G
Na+ channel sequence but lacking
to external TEA also
drkl ~ ~ F W . W ~ T I ~ T ~ T T V G Y G D I Y P - increase
K ~ T Lsensitivity
G G
in slo. Members of the following
occur
naturally
at
the
equivalent positions in
groups of amino acids were considS h d E@FHWAGI;~.MT~T,V.GY G D I C P T T A L G
k
ered to be conserved: [M, I, L, V];
lsl
slo
sequence
(24,
25). For example, the
the
sham L ~ L ' w . w ; ~ -L -.~ ~ ~ ~ - ~ s . v + G Y G D N A P ~ ~ T . Y I G
[A, GI; [S, TI; [Q, Nl; [K, Rl; [E,
presence
of
tyrosine
at
position 292 would
s h d A -,
~- W T YI V ~ ~ ~ T T L G Y (
Dl; [F, Y, W]. (B) Alignment of
I
I T I A G .
be
predicted
to
result
in
a high sensitivityof
H5 domains of K+ channels in the
I,, to external TEA blockade (25). These
Sh superfamily (4, 21), eag (27),
properties have not yet been empirically
and slo.
used it as a probe to isolate the overlapping slo cDNA (Fig. 3) is 4232 nucleotides (16)
cDNAs, 254 and CH4. Although none of with an open reading frame (between posithese cDNAscontains a completeopen reading tions 1 and 3552) that encodes a deduced
frame (ORF), together they yield a composite polypeptide at 1184 amino acids (Fig. 4).
sequence capable of speafylng a polypeptide This sequence appears incomplete at the 5'
over 1100 amino acids in length (Fig. 3A). end (17) but complete at the 3' end because
These cDNAs represent a spliced transcript the untranslated tail of CH4 terminates in a
encompassingover 40 kb of genornic DNA as run of adenine residues preceded by the
polyadenylation signal AATAA (18).
indicated by Southern analysis (Fig. 1B).
Hydropathy analysis (19) revealed seven
The nucleotide sequenceof the composite
-
''
.. .
I
-
$
r
~
I'i
ST
2
s
-
2 AUGUST 1991
1
r
J
REPORTS
553
determined in Drosophila.
Outside these two discrete regions of rimilarity, the Slo sequence is distinct from all
known K+ channels (29). One unusual feature of Slo is its length of 1184 amino acids,
including a stretch of about 850 amino acids
between the S6 domain and the COOHterminus, which contains two additional POtentid
membrane-spanning domains.
Among other K+ channels, only Eag (27)
has a similar length (1174 amino acids).
Across the entire hydrophobic core of the
polypeptide, Slo shares less than 20% amino
acid identity with known K+ channel sequences. In contrast, members of the extended Sh family share at least 35% identity
in the core region (4). These
define Slo
as a
type
P ~ ~ Y P
Because the K+ current affected by slo
requires intracellular
for
we
searched the Slo sequence for an EF hand
that would bind Ca2+ (30). A candidate
domain is present near the COOH-terminus
(Fig. 4). Although this segment deviates
significantly from the consensus EF hand
sequence overall, there is very good agreement with the consensus sequence for those
residues required for coordinating Ca2+
(30). A sequence that matches the adenosine
triphosphate (ATP)-binding site of tyrosine
(31) is located in a neighboring
region (Fig. 4). Although its effect in Drosohas not been characterized, ATP may
directly modulate ca2+-activated K+ thannels in mammalian smooth muscle (32).
sequence
comparisonsamong slo c
~
indicates that the slo transcript is alternatively spliced (Figs. 3 and 4). Alternative splicing of the Sh
generates a number
of gene products with different functional
properties (33). whether alternative splicing
has similar
significance is unknown.
The mutant phenotype that results when
the slo polypeptide is absent or altered allows us to infer the in viva function of the
w i l d - t ~ ~gene
e product' Mutations such as
do4 cause the selective and complete elimination of I ~ , , indicating that the
polypeptide is essential for the expression or
function of the channels mediating this current' We 'Ioned the 'lo locus
On the
basis of the chromosomal location of these
mutations and found that it encodes a polypeptide with sequence similarity to known
K+ channels. Therefore, on the basis of both
functional and structural evidence, we propose that Slo is a strtlctural component of
Ca2+-activatedK+ channels. Together with
s h (3) and eag (26, 27), the locus defines
a third type of K+ channel structural gene
identified and cloned on the basis of its
mutant phenotype.
Although the organization of Slo resem554
bles that of other K+ channel polypeptides,
its overall sequence differs extensively from
these polypeptides, indicating that the divergence of genes encoding voltage-activated
and Ca2+-activatedK+ channels is ancient.
The strong conservation of the H5 domain
in otherwise divergent channels provides
additional support for the fundamental importance of this segment in K+ channel
function. It should now be possible to use
slo to test emerging models for K+ selectivity and permeation and to explore the molecular basis for Ca2+-dependentgating.
R. Coulson, Proc. Natl. Acad. Sci. U . S.A. 74, 5463
~
~
]
,
]
~
~
~
N
~
Regions showing GC compression were sequenced
with deox~inosinetri~hos~hate
( D m ) . Sequence
analyses were performed with software from the
Genetics Computer Group [J, Devereux, P, Haeberli, 0 . Smithies, Nudeic Acids Res. 12, 387
q
~
~~
~
~~
~
Biolok?'
~ e ~
17, c ~2 8 and
~ 254hcontain segments judged to be
unspliced introns (see Fig. 3A) because of the
~
;$
: :i~ g ~ ei c ~ R ~ Sn.10, ~459 w
quences [s. M O W ~ ~, ~ c l e Acids
(1982)], and sequence comparisons with overlapping cDNAs from which these segments were missing. The untranslated segment at the 5' end of 254
preceding the ORF could also be part of an unspliced intron because the first methionine of the
open reading frame is not precededrby a consensus
REFERENCES AND NOTES
translation start site [D. Cavener, Nucleic Acids Res.
1. B. Hille, Ionic Channels ofExcitable Membranes (Sin15, 1353 (1987)l. Furthermore, unlike lo, other
auer, Sunderland, MA, 1984).
K+ channel polypeptides generally have a stretch of
~ 2.PB.~Rudy,
~ ~Neuroscience
.
25, 729 (1988); R. Latorre,
100 to 250 amino acids between the initiating
A. Oberhauser, P. Labarca, 0 . Alvarez, Annu. Rev.
methionine and the first hydrophobic domain (7). If
Physiol. 51, 385 (1989).
an unspliced intron is present at the 5' end of 254,
3. A. Kamb, L. E. Iverson, M. A. Tanouye, Cell 50,
it may include some of the initial sequence present in
405 (1987); D. M. Papazian, T. L. Schwarz, B. L.
the conceptual translition (Fig. 4); several
. Temple, Y. N. Jan, L. Y. Jan, science 237, 749
splice sites occur between the start of the O m and
(1987); 0.
et
E M B O J . 7, 1087 (1988).
the S1 domain. A high frequency of unspliced
4. B. Rudy, C Kentros, E. Vega-Saenz de Miera, J.
introns in c ~encoding
~ ah
~
~
~N ~~than+ ~
Mol. Cell. Neurosci. 2, 89 (1991).
nel has been noted [K. Loughney, R. Kreber, B.
5. L. SalkoK Nature 3023 249 (1983).
Ganeaky, Cell 58, 1143 (1989)l.
6. T. Ekns, B. Ganemky, C.-F. Wu, Proc. Natl. h a d .
18. N. J. Proudfoot and G. G. Brownlee, Nature 263,
Sci. U . S . A . 83, 8415 (1986).
211 (1976).
7. S. Singh and C.-F. Wu, Neuron 2,1325 (1989); A.
19, J. Kyte and R. F. Doolittle, J . M O ~Biol.
.
157, 105
Komatsu, S. Singh, P. Rathe, C.-F. Wu, ibid. 4,313
(1982).
(1990).
20. W. A. Catterall, Science 242, 50 (1988); W. Stuh8. B. S, Pallotta, K. L. Magelby, J. N. Barrett, Nature
mer et al., Nature 339,597 (1989); D. M. Papmian,
293, 471 (1981); R. Latorre, C. Vergara, C.
L. C. Timpe, Y. N. Jan, L. Y. Jan, ibid. 349, 305
Hidalgo, Proc. Natl. Acad. Sci. U . S . A . 79, 805
(1991).
(1982); C. Miller, E. Moczydlowski, R. Latorre, M. 21. L. y . jan and y. N. J ~ ibid.
, 345, 672 (1990).
Phillips, Nature 313, 316 (1985).
22. R. MacKinnon, L. Heginbotham, T. Abramson,
and C.-F. Wu, J. Neurosci. 11, 2135
9. M.
Neuron 5, 767 (1990), H. A. ~ a m n a n net dl.,
(1991).
Science 251, 942 (1991).
10. N. S. Atkinson, G. A. Robertson, B. Ganeuky, Soc.
23. G. Yellen, M. E. Jutman, T. Abramson, R. MacKinNeurosci. Abstr. 15, 541 (1989); G. A. Robertson,
non, Science 251, 939 (1991).
S. Atkbon, B. Ganemky, ibid. 16,670 (1990); 24. A. J. Yo01 and T. Schwarz, Nature 349,700 (1991).
~N.
~
s
,
Biophys. J . 59, 196a (1990).
25. R. MacKinnon and G. Yellen, Science 250, 276
11. D. L. Lidsley and E. H. Grell, ~ e ~ t Variations
i c
~j (1990).
Drosophh melawater (PublicationNo. 627, Camegie
26. R. Drysdale, J. Warrnke, R. Kreber, B. Ganetsky,
Institution of Washington, Washington, DC, 1968).
G e n e t b 127, 497 (1991).
1% To isolate new $0 mutations, wild-cype Canton-S 27, J. Warmke, R. Drysdale, B. Ganetzky, Science 252,
males were mutagenized by y-irradiation (4500 R)
1560 (1991); Y. Zhong and C.-F. Wu, ibid., p.
and mated to bar-3 do' females. Among 25,000 F1
1562.
male offspring screened, three new mutations exhib- 28. M. Gorczyca and C.-F. WU, J . Mem. Biol. 121,237
iting the slo paralytic phenotype (6) were recovered.
(1991).
The presence of bar-3+ identified the mutagenized
29. One additional region of similarity is the alignment
chromosome.
of the S6 hydrophobic domain of Slo with S5 of
13. The electrophysiological phenotype of the new slo
other Kt channels. For example, when aligned with
mutations was assayed by recording action potenthe ~5 segment o f ~ there
h
are ten identities and five
tials in adult flight muscles in response to stimulaconservative substitutions among 21 residues. In
tion of the motor pathway and direct current injeccontrast, the S5 with S5 alignment yields five idention (6).[T. E k n s and B. Ganeuky, J. Neurosci. 8,
tities and three conservative substitutions and the S6
428 (1988)l. The defect in I,, was confirmed by
with S6 alignment yields four identities and four
voltage-clamp analysis of larval muscles for each new
conservative substitutions. The significance of this
slo mutation either when homozygous or in hetalignment between hydrophobic domains that do
eroallehc
withs104,
not occur at corresponding p~sitionsis not known.
14. E. Knust, K. Tietze, J. A. Campos-Ortega, E M B O
30. R. M. Tufty and R. Kretsinge?, s c i e p 187, 167
J . 6, 4113 (1987).
(1975); R. H . Kretsinger, Cold SpringHnrb. Symp.
15. W. Bender, P. Spierrer, D. S. Hogness, J. Mol.
Quant. Biol. 52, 499 (1987).
Biol. 168, 17 (1983).
31. The consensus sequence for the ATP-binding site in
16. The composite cDNA was assembled as follows:
tyrosine kinases is G-X-G-X-X-G-(X)15.20-K[M. P.
nucleotide position 1to 744 (encoding amino acids
Kamps, S. S. Taylor, B. M. Sefton, Nature 310, 589
1 to 248) is from cDNA 254; nucleotide position
(1984)l.
745 to 1626 (encoding amino acids 249 to 542) is
32. C. H . Gelband, S. D. Silberberg, K. Groschner, C.
from cDNA 28; nucleotide position 1626 to 4232
van Breemen, Proc. R . Soc. London Ser. B 242, 23
(encoding amino acids 543 to 1184) is from cDNA
(1990).
CH4. Nucleotide position 1is the first nucleotide of
33. T. L. Schwarz, B. L. Tempel, D. M. Papazian, Y. N.
the open reading frame (ORF) in cDNA 254. The
Jan, L. Y. Jan, Nature 331, 137 (1988); L. C.
ORE of 254 is preceded by a nontranslatable segTimpe, Y. N. Jan, L. Y. Jan, Neuron 1,659 (1988).
ment lacking a consensus translational start signal. 34. Chromosomal in situ hybridization was performed
The ORF of C H i~s followed by an untranslated
as described [W. Engels, C. Preston, P. Thompson,
segment of 680 nucleotides terminating in a
W. Eggleston, Focus 8, 6 (1986)] except that we
poly(A)+ tail. DNA was sequenced in M13 and
used sulfonated (Chemiprobe kit, FMC BioProdpBluescript (Stratagene) vectors by the dideoxy
ucts) rather than biotinylated probes.
chain termination method [F. Sanger, S. Nicklen, A.
35. Consensus recognition sequences were (R,K)-
.
SCIENCE, VOL. 253
~
~
h
~
(R,K)-X-S a (R,K)-(R,K)-X-XSfor adenosine 3',
J.
for &
doacs;I 4 months of age) with a slower -ion,
(CAMPT
m T. Schwuq L.JW Y.N. JW B. HvniLaS
allowe,
he remaining
E.b.
D. 1. Gnva. E.Eeltamm
udE.M~forawidioplikaria:udour
G. ='I.
id. dr;m '252. & 1 1 h 1 ;nd
~o~tl~ucs&dvicclod&mthc&-it~
sixfbmderanimak.whichwereasvmDaJmatc.
(s,,T)-x-(&
for protan &'c
( P K + ~ c & ~ I SUPF%UI
~
by NIH
NS15390. ~
~a&-t did
y nat
transgene (1;).
phosphoryhnon
R Woodgq K. L. Gould, T.
nabk Tnag ud a Khguma EcIkdup to B.G.
Ofthe
Hunter, Eur. J. Biodwrr 161,177 (1986), A K d h N.SA raarvcd suppart 6um NIH gram T32 (RNA)-h
m cr d. I. EM. Ckm 260,12492 (19891.
GMOn31udchcM~mbrDystrophyAssochtioa OftheaarrseeneRvealedRNAsofsizescom.GAR. .& s u p p a t h . &
d w , ~ e a t~ t o v a ~ ~ a n d t o ~ s
36. M.NO&-& d.,Nahtn 320; 188 (1986).
ofW~NIHgrantT32GMOn31,
37. We dedicate this papa to the mcmory of Tom
- , - O D U C . ~ Y ~ ~ ~ M - ~
mmicrnRNAon1yinthehebrainandinthe
s n d i a provided the groundwork for this uulysi.
mbcr3194~~LabornayofGaucia,Uniofd
with disease .yqnp
k t y dWWe thank C.Pemscn, and R Krebcr for tcdmid
toms
(Fig.
1B).
usisancc;F.Blarmcr, L. Mur, and manbas of*
In all animals expressing either construct,
BLmKt Lb for hdp with ~cqutncing;E.bust and
3 May 1991; mcpDd 18 June l99l
- :5
vlon slm IB.
the
u.
*
or&
~~~~Manhp~~w
the pathological findings were resmcted to
the central nervous system (CNS) and to the
striated muxle. The disease phenotype was
transmitted to the offspring with a penetrance
of 100%. ~ n i m kG t h the c k d
Progressive Encephalopathy and Myopathy in
symptoms described above showed variable
Transgenic
HUIMll Foemy vhUSGenes dof selective nerve cel degeneration
in the forebrain, with tissue atrophy and
KATRIN BOTHE, ADRIANOAGUZZI,HANS
LASSMANN,
reactive gliosis. The areas affected most fieAXEL RETHWILM,
IVANHORAK*
quently were the CA3 layer of the hippocampus and the telencephalic cortex (Fig.
Transgenic mice carrying the bel region of human foamy retrovirus (HFV) under 2). Some lesions exhibited a more abnormal
tramaiptiond contrd of its own long terminal repeat expressed the transgene in their pattern, with incomplete cortical and subcentral nervous systems and in smooth and striated muscle tissues. The animals cortical necrosis and occasionally with macdeveloped a progressive degenerative disease of the central nervous system and of the rophages and mild lymphocytic infilaates in
spaces
striated musde. Because -ion
of the transgene was closely correlated with the the meninges and in the periv&
v
c
e of structuraldamage and ' "
ztory reactions were scanty, the disease (13). In situ hybridization analysis revealed a
is likely to be caused dkctiy by the HPV proteins. These unexpectd hdiags caLl for complex pattern of expression in the CNS,
with groups of neurons, oligodendrocytes,
a reevaluation of the pathogenic potential of HFV in humans.
and amocytes containing high amounts of
FV IS A RETROVIRUS ORIGINALLY form becaw of a frameshift mutation in the transcript in the brain and spinal cord. The
isolated 20 years ago from individ- integrase domain that created a premature distribution of positive cells was bilaterally
uals with various diseases (1, 2). stop codon in pol, and (ii) pAgpe, in which symmetrical. In animals 4 weeks old, most
Although the prevalence of HFV was re- 2.3 kb of gagpd and 1.6 kb of env were neurons in the CA3 sector of the hippocamported to be high in certain geographical deleted (7) (Fig. 1A). In both conmuas, pus and a subpopulation of cortical neurons
areas (3), it has not been possible to identify arpression is controlled by the HFV long ter- were strongly positive for HFV RNA, even
HFV as the causative agent of any human mind repeat (LTR).Eight and nine fbunBer in animals in which onset of symptoms had
disease (4). As with the human T cell lym- animals (11) were derived with p W M and not yet ocax~ed(Fig. 2A). In sick animals
phoma virus (HTLV) and human immpAgpe, m q x d d y . F i pHFVM and six older than 6 weeks, most CA3 neurons had
ficiency virus (HIV),the HFV genome has, in pAgpe mice developed a m l o g i c a l syn- degenerated (Fig. 2B), and the highest levels
drome consisting of ataxia, spastic tetraparesis, of expression were seen in cortical neurons.
addition to struaural reaoviral genes, a
containing t h m open redmg frames, M-1 to and blindness. The symptoms in the pHFV,
Two pHFVM and four pAgpe mice
M-3. Bel-1 has some homology to HIV-2 Tat mice appeared as early as 5 to 6 we& after showed focal degeneration of striated mus, led to death
proteinand,simibrfytoHIVTatandHTL.V-I birth, progressed r a p ~ U ~and
Tax,fimaim as a tramaipional mmaaiw- within 4 to 6 we&. The pAgpe transgenic
mice displayed a later onset ofthe disease (1 to
(5-8).
A
Transgenic mice (9) have proved useful in
P
r
?
i
4
=
G
7
8
9
r
O
12
kb
the dissection of the pathogenic potential of
1. (A)
or
and
mlgas
IPO~
1lenv
be1 I I fl
the regulatory genes of the human retrovi4
m~lwqaaon
ma Expressing
H
won
--
rusesHTLV-IandHIV(lO).Toanalyzethe ~ h a v c b e e n s h o w n m ~ H F V
biological activity of HFV regulatory proteins we generated transgenic mice with two
constructs: (i) p w ~ which
h
contains the
complete HFV genome in a noninfectious
-
362isaansgcnicforpHFVM.LVKs1~9~
fiornmousc75:lancl,brain;lant2,liva,lanc3,
~ . ~ a h g ~ ~ a h m ~ . ~ o n t . l n r n
h&
- o lfa n
~cif k r g l a n t 5 , % m u = ; l a n t 6 ,
and hmmnob~
u~vcrsityof wunburg,vaslane 7, musde, lane 8, lymph nodes, lane
7,%
Wunburg, Federal Republic of 9, spkaL Lane 10, mousc 362 bnin; lane 11,
-Y.
w,
w,
~~
mousc253e,lanc12,mousc253brain;lanc
A.
re^^ In*mte of
Pat*,
267 +lane 14,
13,
Dr. Bohr Gasse 7,1030 V i A&
H. Lasmam, h n m of Neurology, Schw~fppanicr- lmarnatebrain;lane15,nontransgauclittamatc brain, lanc 16, mouse 881 forrbrain,h
sassc 17,1090 Vienna, Austria.
17, mouse 881 p a k d arid ,
lane 18,
*To whom a~tspondaKcshould be addrcsscd.
mouse 881 meWhun and brain smn.
2 AUGUST 1991
AI
in cuhmcd cellsAf, Afl
E,
Eco RI,H, Hind Hp, Hpa II,N,Nco I; Pv,
n. (B)
invrau
ti^^ of aansgcnic micc. Micc 75, 253, 267, 11 0 k b
and 881 harbor the p+ tramgene, arid morsc 7 1 k b p.
6
p 5 k b
1
1
v
p
PV
PAgPe
H-E
t
i
!-Ias
M**m
1 2 3 4 5 6 7 8 9 1011 12131415
161718
P-actln
REPORTS 555
~ b g e -