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First published online October 21, 2005
Journal of Experimental Biology 208, 4167-4179 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01857

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Calcium-activated potassium channel of the tobacco hornworm, Manduca sexta: molecular characterization and expression analysis

Matthew R. Keyser and Jane L. Witten^*

Department of Biological Sciences, PO Box 413, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA

View larger version (5K): [in a new window]   Fig. 1. Schematic of strategy used to isolate Manduca slowpoke cDNA. Degenerate oligonucleotide PCR primers designed to conserved regions of the Slo protein were used to amplify cDNA fragments msslo A-1, A-2 and A-3. Each of these cDNA fragments varied slightly in size due to alternate splicing. Manduca sexta specific PCR primers were used in conjunction with degenerate PCR primers upstream and downstream of the A fragments to amplify cDNA fragments msslo B and msslo C. 5' and 3' RACE was performed to attain and confirm the ends of the cDNA sequence. When assembled, an msslo cDNA containing alternate exons E1, G1 and I1 totals 3693 bp in length.

View larger version (109K): [in a new window]   Fig. 2. Deduced amino sequence of msSlo and its alignment to insect and mammalian Slowpoke proteins. The sequences were compared using AlignX software (Informax, Inc.). The black shading indicates identical residues; the gray shading indicates similar residues. The dashes indicate gaps in the alignment. The approximate location of the seven membrane-spanning regions (S0-S6), the pore region (P), the four hydrophobic C-terminal cytoplasmic regions (S7-S10) and the calcium bowl are marked by black bars. The beginning of the five alternate splice sites (A, C, E, G, I) found in the insect sequences are marked with an arrowhead. GenBank accession numbers for sequences: Manduca sexta Slo (AY644784), Drosophila melanogaster Slo (M96840), Periplaneta americana Slo (AF452164), Mus musculus Slo (NM010610). Abbreviations: dSlo, Drosophila melanogaster Slowpoke; msSlo, Manduca sexta Slowpoke; pSlo, Periplaneta americana Slowpoke; mSlo, mouse Slowpoke.

View larger version (32K): [in a new window]   Fig. 3. Alternative RNA splicing of some of the msslo gene products. (A) Schematic diagram of the Slowpoke protein. The hydrophobic core (S0-S6), S4 voltage sensor (+) and hydrophobic tail (S7-S10) containing the RCK domain and calcium bowl are evolutionarily conserved among Slo proteins. Approximate locations of the five alternate splice regions within the tail region that are conserved among insects (A, C, E, G, I) are indicated by black boxes. (B) Alternative forms for msslo regions E, G, I. While some of the exon sequences are conserved among insects, those that are unique to Manduca sexta are denoted with an asterisk (*).

View larger version (36K): [in a new window]   Fig. 4. Developmental profile of msslo mRNA within the CNS. No dramatic changes in msslo gene expression are detected within the CNS during metamorphosis. Total RNA (1 µg per lane) was isolated from day 2 fifth instar larvae (V2), days 0 and 3 wandering larvae (W0, W3), days 0, 2, 4, 6, 8, 10, 12, 14 after pupation (P0, P2, P4, P6, P8, P10, P12, P14) and pharate adults (PA) and hybridized to the digoxigenin-labeled msslo 680 bp cDNA insert or loading control M. sexta ribosomal protein S3 (rpS3) cDNA insert (Jiang et al., 1996). The sizes (kb) of the hybridizing RNAs are shown on the left. The major mRNA is 4 kb and is similar in size to the msslo cDNA. The 11 kb band is probably pre-processed msslo mRNA.

View larger version (11K): [in a new window]   Fig. 5. Semi-quantitative analysis of msslo mRNA developmental profile within the CNS. Densitometric analysis of CNS northern blots using GeneTools software showed relatively constant levels of msslo transcripts throughout larval life, with levels fluctuating during the larval-pupal transition and increasing in adults. Semi-quantification of the chemiluminescence was done by comparing the intensity of the msslo mRNA band with that of the internal control, rpS3. Mean and S.E.M. are plotted (N=5). Abbreviations: day 2 fifth instar larvae (V2), days 0 and 3 wandering larvae (W0, W3), days 0, 2, 4, 6, 8, 10, 12, 14 after pupation (P0, P2, P4, P6, P8, P10, P12, P14) and pharate adults (PA).

View larger version (52K): [in a new window]   Fig. 6. Muscle-specific developmental changes in msslo expression occur in the heart and midgut. (A) In the heart, msslo mRNA expression appears to transiently decline in pupae from larval levels and then return to similar expression levels in pharate adults. (B) In contrast to the heart, midgut levels of msslo mRNA are highest in pharate adults. Total RNA (5 µg) of day 2 fifth instar larvae (V2), day 6 pupae (P6) and pharate adults (PA) was hybridized to the digoxigenin-labeled msslo 680 bp cDNA insert. For the loading control, the lower portion of the blots was hybridized to the digoxigenin-labeled M. sexta ribosomal protein S3 (rpS3) cDNA insert (Jiang et al., 1996). The sizes (kb) of the hybridizing RNAs are shown on the left.

View larger version (11K): [in a new window]   Fig. 7. Semi-quantitative analysis of msslo mRNA developmental profile in visceral muscle. Densitometric analysis of heart (A) and midgut (B) northern blots using GeneTools software confirmed muscle-specific developmental profiles. (A) In the heart, msslo transcript levels transiently decline during development whereas (B) in the midgut, levels are low until the adult stage. Semi-quantification of the chemiluminescence was done by comparing the intensity of the msslo mRNA band with that of the internal control, rpS3. Mean and S.E.M. are plotted (N=3). Abbreviations: day 2 fifth instar larvae (V2), day 6 after pupation (P6) and pharate adults (PA).

View larger version (45K): [in a new window]   Fig. 8. Regional-specific expression of msslo in dorsal longitudinal muscles (DLM). (A) Message levels of msslo in the abdominal DLMs are similar in larvae and pharate adults and are higher than in early-stage pupae. (B) By contrast, transcript levels are dramatically increased in the adult thoracic DLM in comparison with larval msslo mRNA levels. These changes are temporally correlated to the switch in the contractile properties of the thoracic DLMs from tonic in larvae to phasic in adults. Total RNA (1 µg per lane) was isolated from abdominal and thoracic DLMs from day 2 fifth instar larvae (V2), day 6 pupae (P6) and pharate adults (PA) and hybridized to the digoxigenin-labeled msslo 680 bp cDNA insert or loading control M. sexta ribosomal protein S3 (rpS3) cDNA insert (Jiang et al., 1996). The sizes (kb) of the hybridizing RNAs are shown on the left.

View larger version (11K): [in a new window]   Fig. 9. Semi-quantitative analysis of msslo mRNA developmental profile in skeletal muscle confirms regional-specific expression patterns. Densitometric analysis using GeneTools software revealed (A) similar and low levels of msslo transcripts in larval and adult abdominal DLMs compared with early-stage pupae. (B) The dramatic increase in msslo expression in thoracic DLMs is in contrast to the abdominal pattern. Semi-quantification of the chemiluminescence was done by comparing the intensity of the msslo mRNA band with that of the internal control, rpS3. Mean and S.E.M. are plotted (N=5). Abbreviations: day 2 fifth instar larvae (V2), day 6 after pupation (P6) and pharate adults (PA).

View larger version (30K): [in a new window]   Fig. 10. Developmental profile of msslo expression in the thoracic dorsal longitudinal muscles (DLM). Small fluctuations in msslo mRNA levels occur during the transition from the feeding (V) to wandering (W) larval stages and from W to pupal ecdysis (P0). Transcript expression remains low in the early pupal stages (P1-P6), with a moderate increase occurring at pupal days 8-10. The massive upregulation of msslo expression occurs late in adult development on day 15 and appears to gradually decline 2 days after the adult emerges. Total RNA (1 µg per lane) was isolated from thoracic DLMs from days 0 and 2 fifth instar larvae (V0, V2), days 0, 2 and 3 of wandering larvae (W0, W2, W3), and days 0, 2, 4, 6, 8, 10, 12, 14 and 15 after pupation (P0, P2, P4, P6, P8, P10, P12, P14, P15), pharate adults (PA) and in adults just after they emerged and 2 days after emergence (A0, A2). The RNA was hybridized to the digoxigenin-labeled msslo 680 bp cDNA insert or loading control M. sexta ribosomal protein S3 (rpS3) cDNA insert (Jiang et al., 1996). The sizes (kb) of the hybridizing RNAs are shown on the left.

View larger version (20K): [in a new window]   Fig. 11. Temporal correlation of changes in msslo expression with the development of the dorsal longitudinal flight muscle. The dramatic increase in msslo mRNA occurs late in adult development, preceding the muscle's competence to respond to the mature flight motor pattern. The slight increase in message at P8 precedes the first time DLM muscle potentials can be recorded and the onset of maturation of the flight motor pattern within the CNS. Semi-quantification of msslo mRNA was done by comparing its signal intensity to the signal intensity of the housekeeping gene rpS3 using image analysis software (GeneTools). Mean and S.E.M. are plotted (N=5). Developmental events based on Kammer and Kinnamon (1979), Rheuben and Kammer (1980), Duch et al. (2000) and Duch and Levine (2002).

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