Robert Brenner

Molecular Separation of Two Behavioral Phenotypes by a Mutation Affecting the Promoters of a Ca-Activated K Channel

The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations inslowpoke 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) has been identified that removes the neuronal promoters but not the muscle–tracheal cell promoter. This deletion complements the flight defect ofslowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that theash218 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.

Calcium-activated potassium channel gene expression in the midgut of Drosophila

The slowpoke gene of Drosophila encodes a pore-forming subunit of a BK-type Ca(2+)-activated K+ channel. The gene is expressed in neurons, muscles, tracheal cells and in the midgut. The P1 transgene gene contains the entire slowpoke transcriptional control region and drives the expression of a reporter protein comprised of slowpoke amino terminal sequences fused to beta-galactosidase. Here we show that midgut expression is limited to the copper cell and iron cell regions. The copper cell region is composed of two cell types, the copper cells and the interstitial cells. The P1 transgene is expressed in the interstitial cells but not the copper cells. Furthermore, we show that the reporter protein is apically localized in the interstitial cells. In these cells, the slowpoke Ca(2+)-activated K+ channel is thought to participate in the transport of ions between the hemolymph and the lumen of the gut. Subcellularly localized BK channels may be involved in the secretion of acid into the gut lumen. An analogous role for basolaterally localized BK channels has been proposed in the acid-secreting intercalating cells of the human kidney.

Behavioral and electrophysiological analysis of Ca-activated K-channel transgenes in drosophila

Abstract: The slowpoke gene of Drosophila melanogaster encodes a Ca‐activated K channel. This gene is expressed in neurons, muscles, tracheal cells, and the copper and iron cells of the midgut. The gene produces a large number of alternative products using tissue‐specific transcriptional promoters and alternative mRNA splicing. We have described in great depth how transcription is regulated and are now cataloging the tissue‐specificity of different splice variants. It is believed that the diversity of products serves to tailor channel attributes to the needs of specific tissues. Electrophysiological and behavioral assays indicate that at least some of these products produce channels with distinct properties.

Novel embryonic regulation of Ca2+-activated K+ channel expression in Drosophila

Theslowpoke gene ofDrosophila melanogaster encodes a Ca2+-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region ofslowpoke 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. Here we describe and contrast the regulation of neuronal and muscle expression during embryogenesis 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.

Novel embryonic regulation of Ca(2+)-activated K+ channel expression in Drosophila.

Theslowpoke gene ofDrosophila melanogaster encodes a Ca2+-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region ofslowpoke 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. Here we describe and contrast the regulation of neuronal and muscle expression during embryogenesis 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.

Novel embryonic regulation of Ca2+-activated K+ channel expression inDrosophila

Theslowpoke gene ofDrosophila melanogaster encodes a Ca2+-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region ofslowpoke 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. Here we describe and contrast the regulation of neuronal and muscle expression during embryogenesis 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.

β1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The β1 subunit increases Ca2+ and voltagesensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the β1 subunit includes the observations that murine models with deletion of the β1 subunit are hypertensive and that humans with a gain-of-function β1 mutation are at a decreased risk of diastolic hypertension. However, whether the β1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel β1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the β1 subunit ( Kcnmb1-/-) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1-/- mice compared with Kcnmb1+/+ mice, without increased vascular remodeling. β1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of β1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared with Kcnmb1+/+ PASMCs. Compromised PASMC β1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.