The electrical properties of a cell are produced by the complement of ion channels that it expresses. To understand how ion-channel gene expression is regulated, the tissue-specific regulation of the slowpoke Ca²⁺-activated K+ channel gene has been studied. This gene is expressed in the central and peripheral nervous system, in midgut and tracheal cells, and in the musculature of Drosophila melanogaster. The entire transcriptional control region has been cloned previously and shown to reproduce the tissue and developmental expression pattern of the endogenous gene. slo has at least four promoters distributed over approximately 4.5 kb of DNA. Promoter C1 and C1c display a TATA box-like sequence at the appropriate distance from the transcription start site. Promoters C1b and C2, however, are TATA-less promoters. C1, C1b, and C1c transcripts differ in their leader sequence but share a common translation start site. C2 transcripts incorporate a new translation start site that appends 17 amino acids to the N terminus of the encoded protein. Deletion analysis was used to identify sequences important for tissue-specific expression. A transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily was used. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. Different sequences have been identified required for expression in the CNS, midgut, tracheal cells, and muscle (Brenner, 1996a).

The range of electrical properties that a neuron or muscle cell can manifest is determined by which ion channel genes it expresses and in what amounts. The Drosophila slowpoke Ca²⁺-activated K+ channel gene has four distinct promoters. The role that a downstream intronic region, called the C2/C3 region, plays in modulating Promoter C1 and Promoter C2 activity is assessed. Promoter C1 and Promoter C2 appear to be responsible for all neuronal and muscle expression, respectively. Transgenic flies were used to determine the expression pattern from each promoter in the presence and absence of the C2/C3 region. Deletion of this region silences Promoter C1 in adult but not larval CNS and causes a substantial reduction in Promoter C2 activity in adult but not larval muscle. The C2/C3 region also activates Promoter C1 in the animal's eye. By placing the C2/C3 region adjacent to a basal HSP70 promoter it has been demonstrated that the region contains elements that can specifically activate a heterologous promoter in the eye and in adult but not larval muscle. These results demonstrate that the C2/C3 region has a important role in regulating slowpoke developmental expression in the CNS and musculature and in regulating eye expression (Brenner, 1996b).

The slowpoke gene of Drosophila encodes a Ca²⁺-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region of slowpoke is contained in 11 kb of genomic DNA. Previous work has identified four different tissue-specific promoters (Promoters C1, C1b, C1c and C2) and sequences that regulate their activity. The regulation of neuronal and muscle expression during embryogenesis is described and contrasted with its regulation during larval and adult stages. Embryonic regulation is fundamentally different. The embryo uses Promoter C1 and a previously undescribed promoter, called Promoter Ce, to drive neuronal expression. The expression patterns of these promoters are distinct. Muscle expression arises from Promoter C2 as in other developmental stages. A downstream intronic region has been shown to contain control elements that modulate promoter activity differently in embryos, larvae and adults. Embryonic CNS expression is not dependent on the intron, however; its deletion has substantial effects on neuronal expression in larvae and adults. In embryonic muscle, removal of the intron eliminates muscle expression even though this deletion does not reduce larval muscle expression (Thomas, 1998).

Transcriptional regulation of the Drosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified that are responsible for slowpoke expression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, small deletions were introduced into the slowpoke transcriptional control region. Using transformed flies, it was asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion is required for promoter activity. 55-, 214- and 20-nucleotide sequences that control promoter activity have been identified. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells (Chang, 2000).

Based on the expression pattern of mutated reporter constructs, muscles could be grouped into four categories. The data indicate that each group differentially regulates Promoter C2. These groups are (1) larval muscle, represented by the larval body wall muscles; (2) adult asynchronous muscle, represented by the DLM and DVM flight muscle; (3) adult synchronous muscle, represented by the pleurosternal, basalare, pterale, and leg muscles; and finally, (4) jump muscle, represented by a single member, the TT muscle (Chang, 2000).

Evolutionary conservation was used as a rational approach for identifying important transcriptional control elements. Easily identifiable conserved blocks exist between the Promoter C2 control regions of D. melanogaster and D. hydei. Additional deletions were used to further cull unimportant from important sequences. The first, called BR17, removes nucleotides -975 to -401, while the second, called EX, removes nucleotides -400 to -62. In conjunction with the previously described P7 deletion, this provides an uninterrupted set of deletions that approach Promoter C2 from the 5' end. BR17 removes weakly conserved sequence and therefore might be expected to have little effect on Promoter C2 activity. Indeed, the BR17 deletion does not alter the muscle or tracheal cell expression pattern. Deletion EX, however, which removes the strongly conserved 55 box, the 4E region, and the 20 box, has a larger effect. This loss silences Promoter C2 in both adult asynchronous muscle and larval muscle groups. Low level expression persisted in most members of the synchronous muscle group. This is the first indication that some muscles differentially regulate Promoter C2 activity. Each conserved region was inserted back into the EX deletion construct and tested for the capacity to reactivate Promoter C2. The P6 construct represents the intact control region. Whereas the 55 box and the 4E region strongly stimulate larval muscle expression, only the 4E region stimulates expression in adult muscle. Promoter C2 is clearly regulated differently in larval and adult muscle (Chang, 2000).

Removal of the intronic region (+416 to +1473) reduces or eliminates expression in most adult flight muscle, but does not affect expression in larvae. This region includes the intron between exon C2 and C3 (downstream of the Promoter C2 tss) and portions of each exon. Unfortunately, this deletion alters the 5'-untranslated region and splicing of the mRNA encoding the reporter and consequently may alter the translatability or stability of the mRNA. Therefore, the loss of expression might not result from impaired transcription but from a change in mRNA stability (Chang, 2000).

The Gal4BII and Gal4B2.1 transgenes address this caveat. The former contains the intronic region in question, while the latter is lacking it. In both, exon C2 is directly tagged with a Gal4 reporter gene. Exon C2 is the first exon expressed by Promoter C2 and is not found in transcripts expressed by any of the other slowpoke promoters. Because the intronic region is downstream of the Gal4 insertion and not part of the reporter gene mRNA, its removal cannot affect message stability. Interestingly, in the Gal4B constructs, removal of the intronic region eliminates expression in adult asynchronous muscles but does not reduce expression in larval muscle. This is a second illustration of the difference in the regulation of larval and adult muscle groups. Expression in larval muscle is independent of the intronic region, while adult DLM and DVM expression is absolutely dependent on this fragment of DNA (Chang, 2000).

Even within the adult, distinct muscle subtypes show different sequence requirements. Adult thoracic muscles may be categorized as asynchronous or synchronous. Asynchronous flight muscles are optimized for generating force and rapid, repetitive, beating contractions. Neural stimulation makes this muscle competent for contraction but does not trigger a contraction. The synchronous muscles have fewer contractile fibers, a more developed SR, and serve to control flight and move the legs. In this subtype, excitation is tightly coupled to contraction. Asynchronous and synchronous muscle regulate Promoter C2 differently. When the C2/C3 intronic region was deleted (Gal4B2.1 construct), a loss of expression in the asynchronous DLM and DVM has been observed. The deletion does not, however, prevent expression in the synchronous pleurosternal, basalare, pterale, and leg muscles. A second, less robust, example of this dichotomy between asynchronous and synchronous muscle is provided by the EX deletion. EX eliminates expression in the asynchronous DLM and DVM but does not completely eliminate expression in the synchronous pleurosternal, basalare, pterale, and leg muscles (Chang, 2000).

A Gal4BII reporter gene provides a final example of muscle subtype regulation. The insertion of Gal4 into exon C2 causes a specific loss of expression in the TT muscle. This is a synchronous muscle that the animal uses to jump during flight initiation. Expression in other muscle types appears unaffected. The conclusion that Promoter C2 is normally active in the TT is based on the expression pattern of seven different reporter gene constructs and is not in question. The insertion must be responsible. In Gal4BII the structure of the message itself has been altered, which might affect the stability or translatability of the mRNA and results in the specific loss of expression in the TT. However, the most parsimonious explanation is that the insertion, which is adjacent to two evolutionarily conserved mef2 motifs, prevents the binding of factors required for expression in the TT but not in the other muscle types (Chang, 2000).

The slowpoke transcriptional control region is complex, containing at least five tissue-specific promoters. This complexity is mirrored in the regulation of a single slowpoke promoter; Promoter C2. The simplest model consistent with the results is as follows. (1) In general, promoter activation in muscle involves E boxes located in the flanking 4E and intronic regions. These may coordinate the binding of a muscle-activating transcription factor belonging to the myoD basic-helix-loop-helix superfamily. Adult tergotrochanter and asynchronous muscle regions have an absolute dependence for both regions. In larval body wall muscle, however, the intronic region is not required and the requirement for the 4E region can be supplanted by the 55 box. (2) Tracheal cell expression is not absolutely dependent on either of the E box regions that stimulate muscle expression. However, expression in these cells also employs a redundant system requiring the presence of either the 55 or the 20 boxes. The cis-acting 20 box is proposed to bind a transcription factor that stimulates tracheal cell but not muscle expression. It is therefore more specific than the 55 box. (3) It is possible that the capacity of the 55 box to stimulate expression in two very different larval cell types indicates that it participates in developmental stage rather than in tissue-specific stimulation and that it will enhance expression in any larval cell that does not actively prevent activation. However, it is not uncommon for a single transcription factor binding site to be involved in tissue-specific stimulation of transcription in distinctly different cell types (Chang, 2000).

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 (ash2¹⁸), affecting the adjacent gene, has been identified that removes the slowpoke 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 ash2¹⁸ 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 (Atkinson, 2000).

While developmental processes such as axon pathfinding and synapse formation have been characterized in detail, comparatively less is known of the intrinsic developmental mechanisms that regulate transcription of ion channel genes in embryonic neurons. Early decisions, including motoneuron axon targeting, are orchestrated by a cohort of transcription factors that act together in a combinatorial manner. These transcription factors include Even-skipped (Eve), islet and Lim3. The perdurance of these factors in late embryonic neurons is, however, indicative that they might also regulate additional aspects of neuron development, including the acquisition of electrical properties. To test the hypothesis that a combinatorial code transcription factor is also able to influence the acquisition of electrical properties in embryonic neurons the molecular genetics of Drosophila was used to manipulate the expression of Eve in identified motoneurons. Increasing expression of this transcription factor, in two Eve-positive motoneurons (aCC and RP2), is indeed sufficient to affect the electrical properties of these neurons in early first instar larvae. Specifically, a decrease was observed in both the fast K⁺ conductance (I_Kfast) and amplitude of quantal cholinergic synaptic input. Charybdotoxin was used to pharmacologically separate the individual components of I_Kfast to show that increased Eve specifically down regulates the Slowpoke (a BK Ca²⁺-gated potassium channel), but not Shal, component of this current. Identification of target genes for Eve, using DNA adenine methyltransferase identification, revealed strong binding sites in slowpoke and nAcRα-96Aa (a nicotinic acetylcholine receptor subunit). Verification using real-time PCR shows that pan-neuronal expression of eve is sufficient to repress transcripts for both slo and nAcRα-96Aa. Taken together, these findings demonstrate that Eve is sufficient to regulate both voltage- and ligand-gated currents in motoneurons, extending its known repertoire of action beyond its already characterized role in axon guidance. These data are also consistent with a common developmental program that utilizes a defined set of transcription factors to determine both morphological and functional neuronal properties (Pym, 2006).