Tissue-specific expression of a Ca(2+)-activated K+ channel is controlled by multiple upstream regulatory elements

Abstract

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, we are studying the tissue-specific regulation of the slowpoke (slo) Ca(2+)-activated K+ channel gene. 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. Here we demonstrate that s/o 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. We used a transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. We have identified different sequences required for expression in the CNS, midgut, tracheal cells, and muscle.

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

“…Three cDNA fragments, mssloA-1 (1065·bp), mssloA-2 (1032·bp) and mssloA-3 (1131·bp), were obtained with the degenerate forward primer MQYHNKA (5Ј-TGCARTAYCAYAAYA-ARGC-3Ј) and the degenerate reverse primer NFHYHEL (5Ј-AGCTCRTGRTAGTGRAARTT-3Ј), which were designed to the S7-S9 region of the channel (Fig.·1). These three fragments share 100% amino acid identity, with the exception of regions corresponding to alternate exon splice sites E, G and I, which are also found in Drosophila slo (dslo) (Adelman et al, 1992;Brenner et al, 1996) and Periplaneta slo (pslo) (Derst et al, 2003) (Fig.·2). A fourth cDNA fragment (852·bp) 3Ј of mssloA, which we called mssloB, was amplified using the forward degenerate primer NFHYHELK (5Ј-AAYTTYCACTAYC-AYGAGCT-3Ј) and the reverse degenerate primer KRYVITNPP (5Ј-ATRCAKTADTGGTTRGGKGG-3Ј).…”

“…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 singlechannel 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.…”

“…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;. 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).…”

Impaired clock output by altered connectivity in the circadian network

“…slo is highly regulated at the transcriptional level, employing several alternative promoters and splice variants (29)(30)(31). To distinguish between a neuronal and/or a muscular defect associated with the strong impairment observed in slo mutants, we evaluated locomotor activity in two transgenic lines that rescue predominantly neuronal [B52H;slo 4 (32)] or muscular [M131;slo 4 (30)] defects associated with the lack of SLO.…”

Usefulness and Limitation of DiBAC4(3), a Voltage-Sensitive Fluorescent Dye, for the Measurement of Membrane Potentials Regulated by Recombinant Large Conductance Ca2+-Activated K+ Channels in HEK293 Cells

“…The striking observation that the knock-out of the b1 subunit of the BK channel (BKb1) in mice results in spontaneous hypertension suggests BK> 1, which is selectively expressed in smooth muscle cells (4), as an excellent target for antihypertensive drugs (5). However, tissue/ region specific distribution of BK channels (6,7) and the pivotal roles of the channel under both physiological and pathophysiological conditions have been pointed out also in the central and peripheral nervous system (8,9).…”