Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel

Abstract

The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations in slowpoke perturb the signaling properties of neurons and muscles and cause behavioral defects. The animals fly very poorly compared with wild-type strains and, after exposure to a bright but cool light or a heat pulse, exhibit a "sticky-feet" phenotype. Expression of slowpoke arises from five transcriptional promoters that express the gene in neural, muscle, and epithelial tissues. A chromosomal deletion (ash2(18)) has been identified that removes the neuronal promoters but not the muscle-tracheal cell promoter. This deletion complements the flight defect of slowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that the ash2(18) chromosome restores normal electrical properties to the flight muscle. This suggests that the flight defect arises from a lack of slowpoke expression in muscle, whereas the sticky-feet phenotype arises from a lack of expression in nervous tissue.

Fig. 1.

Map of the slowpoke transcriptional control region. The rightward-pointing arrows identify the position of five slowpoke transcriptional promoters. The labels immediately below theline identify the tissue specificity of the each promoter as determined by deletion mapping. A,ApaI; B, BamHI;Bg, BglII; C,ClaI; E, EcoRI;H, HindIII; K,KpnI; M, MunI;N, NcoI; P,PstI; S, SmaI;Sp, SpeI; X,XbaI; Xh, XhoI;Y, XmnI; Z,SphI.

Fig. 2.

The ash2¹⁸deletion eliminates expression from neuronal promoters C0 and C1 but not from the muscle–tracheal cell-specific promoter C2. The products of each promoter begins with a unique exon and can be identified by RT-PCR using exon-specific primers. Total RNA fromash2¹⁸/slo⁴transheterozygous, wild-type, andslo⁴ homozygous flies was reverse transcribed, and the resultant cDNA was subjected to the PCR using the exon-specific primers. PCR products were separated in a 2% agarose gel and stained with ethidium bromide. C0,C1, and C2 identify groups of threelanes displaying products amplified using primers specific for exon C0, C1, or C2, respectively. Amplifications performed onash2¹⁸/slo⁴, wild-type, and slo⁴ RNA are identified by a, w, and s, respectively. From left to right, thearrows identify PCR products diagnostic for the presence of mRNAs that include exon C0, exon C1, and exon C2, respectively. Amplification with exon C0 primers produces the 2844 nucleotide band only from wild-type RNA (lanes 1–3). The exon C1-specific primers produce the diagnostic 2406 nucleotide PCR product only from the wild-type RNA (lanes 4–6). Finally, the exon C2-specific primers amplified the diagnostic 2373 nucleotide product derived from exon C2 from bothash2¹⁸/slo⁴and wild-type RNA but not from theslo⁴ RNA. Other bands are nonspecific PCR artifacts. Conditions favoring maximal sensitivity often lead to the concomitant production of spurious bands.

Fig. 3.

Action potentials produced by dorsal longitudinal flight muscles of wild-type, slo⁴homozygous, andash2¹⁸/slo⁴transheterozygous animals at a stimulation frequency of 20 and 33 Hz. Wild-type muscle produces sharp action potentials at all simulation frequencies tested. Muscle homozygous for theslo⁴ mutation initially produces sharp action potentials, but later spikes are of abnormal shape and breadth. The ash2¹⁸ deletion completely complements the slo⁴action potential phenotype. This indicates that theash2¹⁸ deletion produces functionalslowpoke channels in Drosophilamuscle.

Fig. 4.

Broadening of dorsal longitudinal flight muscle action potentials stimulated at 10, 20, 33, and 50 Hz. The half-height width of six consecutive action potentials were measured. Width is expressed in milliseconds. Black,stippled, gray, and whitebars represent an average from wild-type animals (n= 3), slo⁴/+ heterozygote animals (n = 9),ash2¹⁸/slo⁴transheterozygous animals (n = 6), andslo⁴ homozygous animals (n = 10), respectively. The thin line above the bar represents the SEM for each measurement. Ap1, Ap2,Ap3, Ap4, Ap5, andAp6 represents the width of the first, second, third, fourth, fifth, and sixth action potentials, respectively.

Fig. 5.

Column-based flight assay used to measure the relative capacity of flies for flight. Flies are dropped from a vial into the center of an oil-coated cylinder. The falling flies fly from the center and are trapped in the oil. The distance that they fall is correlated with their capacity for flight (Benzer, 1973; Elkins et al., 1986; Green et al., 1986). The column was fractionated, top to bottom, into bins 5 cm in length. The abscissa represents the distance from the top of the column that the flies fell. The ordinate is the percentage of animals assayed. A, Theash2¹⁸ chromosome was tested for its capacity to complement the flight defect associated with theslo⁴ mutant allele. Results from an assay performed using 952 w¹¹¹⁸, 828slo⁴, and 733ash2¹⁸/slo⁴transheterozygotes. B, Ability of theash2¹⁸ chromosome to complement the flight defect associated with theslo¹ mutant allele. Results from an assay performed using 1002 w¹¹¹⁸, 903 w¹¹¹⁸; st bar3slo¹,and 369ash2¹⁸/st bar3slo¹ flies. Thew¹¹¹⁸ stock carries a wild-type copy of the slowpoke gene and served at the positive control. Wild-type flies (squares) and theash2¹⁸/slo⁴orash2¹⁸/slo¹transheterozygotes (triangles) accumulate near the top of the column. The slo⁴ orslo¹ homozygotes (circles) accumulate deeper in the column. Flies that did not initiate flight are not counted and are trapped in a pool of oil at the bottom of the column.

Fig. 6.

An example of the sticky-feet phenotype exhibited by animals homozygous for null mutations in the slowpokegene. This particular homozygous slo⁴male has been exposed to a bright light (see Materials and Methods) for 15 sec and is being pushed with a number 2 pencil. Such animals do not attempt to escape or avoid the pencil and hang onto the surface on which they stand. The animal is shown leaning over in response to the pressure. With continued pressure, the animal will topple over and then in a very uncoordinated manner attempt to right himself. If successful, such an animal will usually once again exhibit the sticky-feet phenotype. Recovery from the behavior can take many minutes and seems to be speeded by repetitive stimulation.