Calcium-activated potassium channel of the tobacco hornworm, Manduca sexta: molecular characterization and expression analysis
Department of Biological Sciences, PO Box 413, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA
* Author for correspondence (e-mail: jlw{at}uwm.edu)
Accepted 24 August 2005
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Summary |
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Key words: Slowpoke, developmental regulation, gene expression, tissue-specific expression
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Introduction |
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The functional diversity of Slowpoke channels is thought to arise from
extensive regulation of a single slo gene and its gene products.
Distinct transcripts can shape the channel properties to the requirements of
the cells, tissue, developmental stage or physiological state. In
Drosophila, tissue-specific transcriptional promoters
(Atkinson et al., 2000;
Brenner et al., 1996
) and
alternative splicing (Adelman et al.,
1992
; Derst et al.,
2003
; Lagrutta et al.,
1994
) generate a large number of transcripts that could modify
channel properties such as single-channel conductance, calcium sensitivity and
mean open time (Adelman et al.,
1992
; Lagrutta et al.,
1994
). In vertebrates, alternatively spliced BK transcripts are
expressed in distinct patterns in the brain
(Tseng-Crank et al., 1994
),
cochlea (Jiang et al., 1997
;
Langer et al., 2003
), kidney
(Bravo-Zehnder et al., 2000
)
and smooth muscle of arteries, esophagus and uterus
(Knaus et al., 1994
;
Salapatek et al., 2002
;
Zhou et al., 2000
), suggesting
isoform-specific functions. Alternate exon selection in mammals can fluctuate
depending upon the physiological state; for example, pregnancy (Benkusky et
al., 2000
,
2002
) and stress
(Xie and McCobb, 1998
). The
functional consequences of reversible post-translational modification of Slo
channels by serine/threonine and tyrosine kinases are complex and can be
isoform- and tissue-specific. For example, protein kinase A (PKA) activates
the ZERO splice variant of BK channels expressed in HEK (human embryonic
kidney) cells but inhibits the activity of the stress axis regulated exon
(STREX) isoform (Tian et al.,
2001
). In glial cells, PKA phosphorylation enhances BK channel
activity whereas protein kinase C (PKC) reduces channel gating
(Schopf et al., 1999
).
Tyrosine kinase phosphorylation of BK channels can lead to smooth muscle
vasoconstriction due to inhibition of channel activity
(Alioua et al., 1998
) while
enhancement of calcium-sensitive gating can occur in heterologous expression
systems (Ling et al., 2000
).
BK channels also are modulated by phosphatases, which can lead to enhanced
channel activity in pituitary tumor cells
(White et al., 1991
) and in
HEK cells by reducing PKA inhibition (Tian
et al., 2001
). In addition, association with beta and other
accessory subunits or binding proteins can profoundly alter channel properties
such as calcium sensitivity and gating
(Xia et al., 1998
;
Zhou et al., 1999
).
Far less is known about how developmental changes in slo
expression levels alter cellular excitability and contribute to synaptic and
behavioral plasticity (Becker et al.,
1995; Brenner et al.,
1996
; Lhuillier and Dryer,
2000
; Muller and Yool,
1998
). Metamorphosis of the tobacco hornworm, Manduca
sexta, provides the opportunity to analyze the effects of changing ion
channel gene expression and channel density in vivo at the level of
identified neurons or muscles whose developmental fates and behavioral roles
are known (Truman, 1992
;
Weeks et al., 1997
).
Developmental changes occur in calcium and potassium currents that could
tailor the electrical properties of neurons, glia or muscles to permit
postembryonic changes in behavior (Duch
and Levine, 2002
; Hayashi and
Levine, 1992
; Mercer and Hildebrand,
2002a
,b
).
However, none of the genes or gene products producing these currents in
Manduca sexta are known. Only one potassium channel gene has been
cloned from this insect: Manduca sexta ether à-go-go, or
mseag (Keyser et al.,
2003
). The ether à-go-go (eag) gene was
isolated from Drosophila melanogaster based on its ether-induced
leg-shaking mutant phenotype (Kaplan and
Trout, 1969
; Ganetzky and Wu,
1983
) and is the prototype of a subfamily of voltage-gated
potassium channels that includes ether à-go-go related
(erg) and ether à-go-go like (elk)
(Warmke and Ganetkzy,
1994
).
Here, we present the isolation of the cDNA that encodes the alpha subunit of a calcium-activated potassium channel, Manduca sexta slowpoke (msslo), and its postembryonic temporal profile in the CNS, visceral muscles and two sets of skeletal muscles. Our data show that msslo gene expression is regulated developmentally in a tissue-specific pattern suggesting that the msSlo currents may contribute to the changes in neural circuits and muscle properties that produce stage-specific functions and behaviors.
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Materials and methods |
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We use abbreviations to indicate the different developmental stages of the
insect: roman numeral V for the final or fifth instar larvae, W for the
wandering stage, P for pupal animals, PA for pharate adults (insects on the
day of emergence but still within the pupal cuticle), and A for emerged
adults. Insects are staged using discrete developmental markers: larval, pupal
and adult ecdysis (V0, P0 and A0, respectively) and the onset of `wandering'
behavior by the fifth-stage larvae (W0). The number following the stage
designates the number of days past the molt. For example, V2 indicates a
larval animal that molted 2 dayspreviously. Cuticular and pigmentation markers
were used to stage developing adults and adults near eclosion
(Schwartz and Truman, 1983;
Curtis et al., 1984
).
Reverse-transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from larval CNS and skeletal muscle and pharate
adult thoracic dorsal longitudinal muscles (DLM) by mechanical homogenization
in TRIzol reagent (GIBCO-BRL, Gaithersburg, MD, USA), pooled, its integrity
confirmed on a 1% non-denaturing agarose gel stained with ethidium bromide,
and quantified by UV spectrophotometry. Complementary DNA was synthesized from
1 µg of total RNA with 200 units of Superscript II reverse transcriptase
(GIBCO) and 150 ng of random hexamer primers. The cDNA reaction was diluted
2.5-fold with double-distilled H2O (ddH2O) in
preparation for PCR amplification. The cDNA fragments were amplified using 0.5
units of Taq polymerase (Promega, Madison, WI, USA) in PCR buffer B (Promega),
pH 8.0, containing 2.5 mmol l-1 MgCl2, 200 µmol
l-1 dNTPs (Promega), 0.4 µmol l-1 of each
Manduca specific primer, 0.8 µmol l-1 of each
degenerate primer (The Great American Gene Company, Ramona, CA, USA) and 1
µl of diluted cDNA as template in a thermocycler (MJ Research, Waltham, MA,
USA) using the following touchdown PCR paradigm
(Don et al., 1991) to increase
specificity: (94°C, 1 min; 65°C-0.8°C/cycle, 2.5 min; 72°C, 1
min) for 19 cycles followed by (94°C, 1 min; 55°C, 1 min; 72°C,
1.5 min) for 35 additional cycles.
Three primer pairs designed to regions of high conservation and low degeneracy between Drosophila melanogaster Slo (dSlo) and Rattus norvegicus Slo amino acid sequences were used to PCR amplify the msslo cDNA. To obtain the large cytoplasmic tail region, a degenerate primer pair was designed to the conserved S7 domain [MQYHNKA (5'-TGCARTAYCAYAAYAARGC-3')] and to the S9 domain [NFHYHEL (5'-AGCTCRTGRTAGTGRAARTT-3')]. The degenerate forward [NFHYHELK (5'-AAYTTYCACTAYCAYGAGCT-3)] and reverse KRYVITNPP (5'-ATRCAKTADTGGTTRGGK GG-3')] primers were designed to a conserved region 3' to the S10 domain. To obtain the S1-S6 domains, we designed a degenerate primer to a conserved region 5' of the S1 domain [KDWAGE (5'-WGYGAARGACTGGGCWGG-3')] and a Manduca specific primer within S7 (5'-TGGGATATTCAGCAGGTAG-3'). All PCR products were separated on a 1.0% agarose gel, cloned into the pGEM-T Easy vector (Promega), and sequenced.
5' rapid amplification of cDNA ends (5' RACE)
Total RNA was extracted from pharate adult thoracic DLM as described above
and reverse transcribed with 150 units of Omniscript reverse transcriptase
(Qiagen, Valencia, CA, USA) primed with the Manduca specific
oligonucleotide primer 5'-GAATGCTACTAGCGAACATTGC-3'. Excess primer
was removed from the cDNA reaction on a MinElute DNA purification column
(Qiagen) prior to modification in a (dATP)n tailing reaction using
terminal transferase (Promega) and diluted fivefold with ddH2O for
PCR amplification. The 5' RACE products were PCR amplified (reaction
components described earlier) by using the adapter primers
GACTCGAGTCGACATCG(T)17 and GACTCGAGTCGACATCG
(Sambrook and Russell, 2001),
designed to the synthesized poly A tail at the 3' end of the cDNA
(5' end of mRNAs), and the specific primer
5'-CACCAACACTACCAGTATTCG-3' under the following touchdown PCR
paradigm: (94°C, 5 min; 55°C, 5 min; 72°C, 40 min) one cycle,
followed by (94°C, 40 s; 58°C - 0.2°C/cycle; 72°C, 3 min) for
35 cycles followed by a final extension at 72°C for 15 min. The PCR
reaction was diluted four-fold and re-amplified using the same set of primers
[minus the (T)17 modified primer] using the following paradigm
(94°C, 1 min; 55°C, 1 min; 72°C, 1 min) for 30 cycles to increase
yield and specificity of the PCR products. The PCR products were separated on
a 1.5% agarose gel, cloned into the pGEM-T Easy vector and sequenced.
3' RACE
PolyA mRNA extracted from 100 µg DLM total RNA using an Oligotex mRNA
purification system (Qiagen) was reverse transcribed with 150 units of
Omniscript reverse transcriptase (Qiagen) and then the oligonucleotide primer
(T)17 diluted fivefold with ddH20 in preparation for PCR
amplification. The 3' RACE products were amplified using the adapter
primers 5'-GACTCGAGTCGACATCG(T)17-3' and
5'-GACTCGAGTCGACATCG-3' and the specific primer
5'-CGACACCTCCTCCTCCTGC-3' under the following touchdown paradigm:
(94°C, 5 min; 55°C, 5 min; 72°C, 40 min) one cycle, followed by
(94°C, 1 min; 68°C-0.6°C/cycle, 1 min; 72°C, 1 min) for 19
cycles and (94°C, 1 min; 58°C, 1 min; 72°C, 1 min) for 35 cycles
with a final cycle of (94°C, 40 s; 55°C, 1 min; 72°C, 15 min). To
increase yield and specificity, the reaction was diluted fourfold with
ddH2O and re-amplified using the nested specific primer
5'-CGACACCTCCTCCTCCTGC-3' and the adapter primer [minus the
(T)17 modified primer] under the same paradigm used to re-amplify
the 5' RACE products. The PCR products were separated on a 1% agarose
gel, cloned into the pGEM-T Easy vector and sequenced.
Sequence analysis
The plasmid clones containing the isolated cDNA fragments were sequenced
using Big Dye technology and ABI Model 377 Prism DNA Sequencers (Foster City,
CA, USA) at the Iowa State DNA Sequencing and Synthesis Facility (Iowa State
University, Ames, IA, USA). Universal vector- and sequence-specific primers
were used to generate overlapping sequence products from sense and antisense
ssDNA strands. The sequencing fragments were assembled using ContigExpress DNA
sequence analysis software (Informax, Inc., Frederick, MD, USA), and amino
acid alignments between Slo family members were made using a modification of
the ClustalW algorithm within the AlignX sequence alignment software
(Informax, Inc.). Consensus sequences for enzymatic modifications were
performed using Prosite (Swiss Institute of Bioinformatics, Geneva,
Switzerland) and NetPhos2.0 (CBS, Technical University of Denmark).
Northern blot analysis
Total RNA (100 µg) was extracted from staged CNS, skeletal muscles and
visceral muscles from 5-15 animals per stage through mechanical homogenization
and two successive extractions in TRIzol reagent. RNA integrity was verified
by non-denaturing gel electrophoresis on an ethidium bromide-stained 1%
agarose gel, and the concentration of RNA quantified by UV spectrophotometry.
RNA samples were adjusted to a concentration of 1 µg µl-1 in
RNA storage solution (1 mmol l-1 sodium citrate, pH 6.4; Ambion,
Austin, TX, USA). 1 µg of CNS and skeletal muscle and 5 µg of midgut and
heart total RNA were separated by electrophoresis on a 1.2% denaturing agarose
gel (1x MOPS and 2.2 mol l-1 formaldehyde), transferred to a
positively charged nylon membrane (Roche, Indianapolis, IN, USA), UV
cross-linked and hybridized to digoxigenin (DIG)-labeled RNA probes.
The RNA probes were transcribed from linearized pBSK plasmid clones
(Stratagene, La Jolla, CA, USA) containing either a 680 bp PCR fragment of
msslo amplified from cDNA using the specific primers
5'-GCCCTTCAAACAGGCTACAGAG-3' and
5'-GACGACCAGTCGAAAGATTTC-3' or containing the coding sequence for
Manduca sexta ribosomal protein S3 (rpS3;
Jiang et al., 1996; generously
provided by Dr Michael Kanost, Kansas State University). The probes were
purified on an RNeasy RNA purification column (Qiagen) and visualized on a
non-denaturing 1.5% ethidium bromide-stained agarose gel. Incorporation rate
of DIG-UTP was assessed by dot blot analysis and was used to estimate probe
concentration. Membranes were hybridized with probes at a concentration of 100
pg ml-1 for msslo and 25 pg ml-1 for
rpS3 in preheated Ultrahybe hybridization buffer (Ambion) overnight
at 68°C in a Little Shot hybridization oven (Boekel, Festerville, PA,
USA). The membranes were washed sequentially at 68°C in 2x SSC/0.1%
SDS for 45 min, 0.5x SSC/0.1% SDS for 30 min, and 0.1x SSC/0.1%
SDS for 30 min. The hybridized probe was detected using anti-DIG:alkaline
phosphatase (1:5000) and its chemiluminescent substrate CDP-Star as described
in the protocol provided by Roche. Exposure time to Biomax light film (Eastman
Kodak, Rochester, NY, USA) varied between 30 s and 10 mindepending on signal
intensity.
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Results |
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Sequence fragments obtained from plasmid clones containing mssloA-C cDNAs and from plasmid clones containing 5' and 3' RACE-generated cDNAs were assembled using ContigExpress DNA sequence analysis software (Informax, Inc). When mssloA-1 was included into the sequence assembly, the fragments generated a 3693 bp nucleotide sequence containing the open reading frame and encoding a 1129 amino acid polypeptide (Fig. 2). The Manduca slo sequence was entered into GenBank (AY644784). Analysis of the deduced Manduca protein revealed conserved domains common to all voltage-gated potassium channels including the six transmembrane domains (S1-S6), the voltage sensor (S4) and a pore lining region between S5 and S6 characterized by the GYG K+ specificity filter motif. Conserved domains specific to large-conductance calcium-dependent voltage-gated K+ channels were also identified, including an S0 transmembrane domain, cytoplasmic hydrophobic segments S7-S10, the regulation of conductance of potassium (RCK) domain-containing sites critical for Mg2+ binding, a calcium bowl, and multiple C-terminal alternative splice regions (Fig. 2).
Five alternative splice sites within the C-terminus, A, C, E, G and I, have
been described within dslo
(Adelman et al., 1992;
Lagrutta et al., 1994
) and
within pslo (Derst et al.,
2003
) as sites for insertion of an array of small exons encoding
different polypeptides. Alignment of msslo cDNA fragments A1-A3,
B and C to dslo and pslo reveals that the
location of each alternate splice site is conserved: site A is within the S6
transmembrane domain, site C is within the RCK domain, site E lies partly
within the S8 domain, while sites G and I are within variable regions between
the S8 and S9 domains (Fig. 2).
Msslo cDNA fragments contained some unique combinations of exons E, G
and I: mssloA-1 contained exons E1 (111 bp; AY644784), G1 (63 bp;
AY644784) and I1 (42 bp; AY644784), mssloA-2 contained exons E2 (111
bp; AY644785), G2 (48 bp; AY644787) and I2 (32 bp; AY644788),
mssloA-3 contained exons E2, G3 (147 bp; AY644786) and I2,
while mssloB contained exons A1 (87 bp; AY644784) and C1 (105;
AY644784) (Figs 1,
2,
3).
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Sequence comparison of the deduced Manduca protein (NCBI, protein BLAST) revealed the highest amino acid identity to insect Slo channels (>80%), fly (Drosophila melanogaster), cockroach (Periplaneta americana) and mosquito (Anopheles gambiae), with less identity to nematode (60%; Caenorhabditis elegans) or mammalian (55%; rat, mouse or human) Ca2+-activated K+ channels. The regions of highest conservation between the insects have the following amino acid identities: segments S0-S6, 93-95%; the RCK domain, 97-98%; and segments S9-S10, 86%. The only region exhibiting low identity to Slo insect family members other than the 5' and 3' ends lies within the cytoplasmic tail region between S7 and S9 (58-64%). Part of the divergence among insect Slo proteins can be attributed to alternate exons E, G and I, which reside within this segment. While exons E1, E2 and G3 are conserved among insects (69-94% identity), exons G1, G2, I1 and I2 are unique to Manduca (0% identity) and could endow msSlo channels with a different range of biophysical properties to suit species-specific behaviors. Thus, the Manduca cDNA that we isolated is likely to be an ortholog of the slo family of calcium-activated potassium channels and will be referred to as msslo.
Msslo gene expression is developmentally regulated in tissue-specific patterns
In Drosophila, transcriptional regulation of slo directs
expression of gene products in neurons and other cell types including muscles,
midgut and trachea at embryonic and postembryonic life stages
(Becker et al., 1995).
Developmental and tissue-specific expression levels are not easily quantified
in Drosophila. We used a highly sensitive non-isotopic northern blot
assay to quantify msslo expression during development in the nervous
and several muscle systems to complement and extend the molecular genetic
analysis in Drosophila. The spatial distribution of the slo
transcript in Manduca was also widespread, but the developmental
expression patterns were tissue specific, with the most dramatic changes
occurring in one set of skeletal muscles.
In all tissues, we detected a major mRNA of 4 kb. The size of this mRNA is similar to the size of the msslo cDNA, suggesting that the cDNA we isolated was full length. A much larger mRNA (11 kb) that may represent pre-processed RNA was observed at a few developmental stages, primarily in the CNS (Fig. 4).
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Developmental changes in msslo expression occur in visceral muscle
Northern blot expression analysis of the visceral muscles, heart and midgut
was performed at three stages of the animal's life: larval on V2, pupal on P6
and in adults on the day of their emergence (pharate adults, PA). We observed
marked muscle-specific developmental regulation of msslo gene
expression in the heart and midgut (Figs
6,
7). In the heart, levels of
msslo mRNA transiently declined in pupae from larval levels then
returned to similar levels of expression in the adult (Figs
6A,
7A). In the midgut, levels of
the transcript are barely detectable in larvae and pupae but are present in
the pharate adult (Figs 6B,
7B). Our hybridization analysis
also suggests that msslo mRNA levels are generally lower at all
stages in the heart and midgut than in the CNS because five times more total
RNA from these visceral muscles was needed for transcript detection (see
Materials and methods).
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Discussion |
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Steroid hormone modulation of ion channel gene expression
Steroid hormone status can dynamically regulate the excitability of
vertebrate neurons, endocrine and muscle cells by affecting the expression
levels, splicing or activity of BK channels. For example, adrenal steroids
downregulate BK channel currents in hippocampal neurons
(Kerr et al., 1989) and can
mediate the inclusion of the stress axis regulated exon (STREX) in adrenal
chromaffin cells (Shipston et al.,
1996
; Xie and McCobb,
1998
; Tian et al.,
1999
). Fluctuations in estrogen and/or progesterone during
pregnancy may influence alternative exon splicing in mouse uterine muscle,
altering muscle contractility (Benkusky et
al., 2000
). The steroid hormone 20-hydroxyecdysone (20HE) mediates
many of the metamorphic changes in the nervous and muscular systems that
reorganize the caterpillar to form adult structures and behavior
(Truman, 1992
;
Weeks and Levine, 1995
).
Steroid-mediated transcriptional control of ion channel gene expression could
alter the electrical properties of larval neurons and muscles to tailor their
electrical properties for new adult circuits and behavior. Developmental
changes in potassium currents occur in Manduca leg
(Hayashi and Levine, 1992
;
Grüenwald and Levine,
1998
), flight motoneurons
(Duch et al., 2000
;
Hayashi and Levine, 1992
),
antennal lobe neurons (Mercer and Hildebrand,
2002a
,b
)
and glia (Lohr et al., 2001
),
but the ion channel genes or gene products producing these potassium currents
are not known yet.
We have isolated and molecularly characterized the developmental expression
profile for the only two voltage-gated potassium ion channel genes isolated
from Manduca sexta: msslo reported here and Manduca
sexta ether à-go-go or mseag
(Keyser et al., 2003). The
changes in expression levels of both genes are temporally correlated to the
fluctuations in 20HE that mediate metamorphosis, suggesting that they may be
regulatory targets of the ecdysteroids. The developmental profiles for each
gene are distinctive and are tissue-specific. For example, when 20HE titers
are increasing early in adult development, CNS levels of mseag
transcripts are highest, while msslo transcripts are low. When
steroid titers have declined prior to emergence, CNS mseag levels are
low, while CNS msslo transcripts have slightly increased and flight
muscle transcripts have dramatically increased. This differential
developmental regulation for msslo and mseag may contribute
to altering the cell's excitability to meet stage-specific needs.
As in vertebrates, steroid-mediated transcription control of msslo exon choice could be another mechanism to modify cell excitability during postembryonic development. Because the size of the alternate exons is relatively small (100 bp) incomparison with the 4 kb transcript, our northern blot analysis would not detect these changes. With single-cell RT-PCR of identified neurons, it will be feasible to investigate whether msslo exon selection is regulated at the cellular level, changes during development and if the exon choice is mediated by the ecdysteroids.
Are developmental changes in msslo expression in visceral muscle related to stage-specific functions?
In the Drosophila heart, four K+ currents, including a
Slo current, have been detected through mutational and pharmacological
analysis (Johnson et al.,
1998). The role that dSlo plays in maintaining heartbeat is
critical, as both slo mutants and animals injected with the agent
charybdotoxin, which blocks fast Ca2+-gated K+ channels,
exhibit greatly diminished heartbeat and rhythmicity
(Johnson et al., 1998
). Given
the importance of the Slo channel in the Drosophila heart, it is
likely that the developmental regulation of msslo expression we
observed in Manduca has stage-specific physiological relevance. For
example, during metamorphosis, there is a switch in pacemaker regions
(Davis et al., 2001
;
Slama, 2003
). In larvae,
peristaltic contractions of the heart are anterograde, while in the adult the
heartbeat cycles between anterograde and retrograde contractions. This change
in pacemaker regions may occur as a result of the heart becoming innervated
during the metamorphic transition (Davis
et al., 2001
; Dulcis et al.,
2001
). Because dSlo channels are critical for pacemaker activity
in Drosophila (Johnson et al.,
1998
), it is possible that developmental regulation of msSlo
channel expression levels or their spatial distribution could contribute to
the change in pacemaker localization.
The midgut is a primary site for nutrient and ionic regulation in insects.
In the midgut of Drosophila, Slo expression is apically localized to
interstitial cells within the copper cell region
(Brenner and Atkinson, 1997).
These cells are thought to be involved with potassium ion transport between
the hemolymph and the gut lumen. An analogous role has been proposed for the
goblet cells in the Manduca larval midgut
(Cioffi, 1979
). During
postembryonic development, the levels of msslo transcript are barely
detected in larvae and pupae and appear to be upregulated in the pharate adult
stage. This upregulation of msslo may be related to differences in
ion transport that accompany a dietary change from feeding on tobacco leaves
to drinking nectar.
Upregulation of msslo mRNA expression in the dorsal longitudinal muscles is correlated with key developmental events
Electrophysiological analysis in Drosophila confirms the presence
of mature Slo currents in the embryonic, larval body wall muscle and adult
DLMs but not in pupal muscles (Elkins et
al., 1986; Salkoff,
1983a
,b
,
1985
;
Singh and Wu, 1990
). The lack
of detectable currents in pupal muscles could be due to very low channel
density since expression levels of the dslo mRNA in these muscles was
thought to be much lower than in the CNS since they could only be detected by
RT-PCR or reporter gene expression (Becker
et al., 1995
; Brenner et al.,
1996
). By contrast, msslo transcripts are easily detected
in northern blot analysis in homologous muscles at all stages and at a higher
level of expression than in the CNS. These results raise the possibility that
functional msSlo currents may be present and participate in early as well as
late neuromuscular development of the flight system.
The first increase in msslo transcript in the DLMs occurs during
the wandering stage and is concurrent with the onset of degeneration of larval
muscle fibers, retraction of the innervating nerve terminals and myoblast
proliferation (Fig. 11)
(Duch et al., 2000). BK
channels can participate in programmed cell death in vascular smooth muscle
(Krick et al., 2001
), and
potassium efflux is a major component in neuronal apoptosis
(Yu, 2003
;
Yu et al., 1997
). If the
msslo channel is functional at this time, it could play a role in
larval muscle fiber degeneration. Another possibility is that the increase in
msslo mRNA and channel density may alter muscle properties to
facilitate pupal ecdysis, which occurs just after the wandering stage.
The next increase in msslo message occurs approximately midway
through adult development (P8-P10), temporally correlated with significant
increases in muscle mass, differentiation, more extensive terminal innervation
and the onset of muscle membrane excitability
(Fig. 11)
(Duch et al., 2000;
Rheuben and Kammer, 1980
).
Nerve-muscle interactions are critical in the development of flight muscles in
Drosophila (Fernandes and
Keshishian, 1998
) and in Manduca sexta
(Bayline et al., 2001
), so if
msSlo currents are present in the developing DLMs, they may play an active
role in these processes.
Upon adult emergence in Drosophila, the inward voltage-gated
calcium current and the Slo current mature, with the latter supplanting the
fast inactivating Shaker current as the repolarizing current
(Salkoff, 1985). That the
msslo mRNA upregulation precedes eclosion by at least 3 days suggests
there is a build-up of gene products that precedes the appearance of the
mature current, as is seen with voltage-gated Ca2+ channels in
developing Drosophila flight muscle
(Wei and Salkoff, 1986
).
Although specific currents have yet to be identified in Manduca
flight muscle, there is a slight reduction in the duration of the adult flight
muscle action potential as compared with the larval one
(Rheuben and Kammer, 1980
).
Upregulation of msslo may contribute to the more rapid repolarization
of the action potential through increased channel density. This change in
electrical properties may be necessary for the performance of the
high-frequency flight motor program
(Kammer and Kinnamon, 1979
).
With the use of RNA interference (Feinberg
and Hunter, 2003
; Uhlirova et
al., 2003
) and electrophysiological analysis, we will be able to
test whether developmental changes in msslo expression contribute to
the remodeling of electrical properties of specific neurons and muscles and
synaptic plasticity.
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Acknowledgments |
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References |
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