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

Abstract

Large-conductance calciumand voltage-gated potassium channels (BK or Slowpoke) serve as dynamic integrators linking electrical signaling and intracellular activity. These channels can mediate many different Ca 2+ -dependent physiological processes including the regulation of neuronal and neuroendocrine cell excitability and muscle contraction. To gain insights into the function of BK channels in vivo, we isolated a full-length cDNA encoding the alpha subunit of a Slowpoke channel from the tobacco hornworm, Manduca sexta (msslo). Amino acid sequence comparison of the deduced Manduca protein revealed at least 80% identity to the insect Slo channels. The five C-terminal alternative splice regions are conserved, but the cloned cDNA fragments contained some unique combinations of exons E, G and I. Our spatial profile revealed that transcript levels were highest in skeletal muscle when compared with the central nervous system (CNS) and visceral muscle. The temporal profile suggested that msslo expression is regulated developmentally in a tissue-and regional-specific pattern. The levels of msslo transcripts remain relatively constant throughout metamorphosis in the CNS, transiently decline in the heart and are barely detectable in the gut except in adults. A dramatic upregulation of msslo transcript levels occurs in thoracic but not abdominal dorsal longitudinal body wall muscles (DLM), suggesting that the msSlo current plays an important role in the excitation or contractile properties of the phasic flight muscle. Our developmental profile of msslo expression suggests that msSlo currents may contribute to the changes in neural circuits and muscle properties that produce stage-specific functions and behaviors.

Steroid hormone regulation of the voltage‐gated, calcium‐activated potassium channel expression in developing muscular and neural systems

“…Previous work using a Northern Blot assay has shown that Manduca sexta slowpoke (msslo) mRNA expression levels in thoracic dorsal longitudinal muscles (DLMs) are low from the fifth instar until P15 when they abruptly increase in the developing adult (Keyser and Witten, 2005). Using the more sensitive qPCR technique, we observed a similar developmental profile, with an abrupt 12.6-fold increase in msslo transcript levels, two-thirds through adult development on P15 (Fig.…”

“…Previous research using Northern Blot analysis revealed differential msslo transcript expression patterns during late adult development in the CNS and the DLMs. Although msslo is abruptly upregulated in the DLMs, it remains relatively low and only gradually increases in the CNS (Keyser and Witten, 2005). Using the more sensitive qPCR technique, we detected a 2.9-fold increase in CNS msslo expression at the PA stage (Fig.…”

“…The ecdysteroid titer decline during late adult development precedes an abrupt upregulation in msslo mRNA expression at P15 (Keyser and Witten, 2005). To test if msslo was a downstream target of the ecdysteroids, we manipulated ecdysteroid levels prior to P15.…”

“…This upregulation at PA also correlates with the ecdysteroid titer decline, suggesting that the hormones may act to indirectly regulate msslo mRNA levels in the DLMs and CNS. Since msslo upregulation follows titer decline in the CNS (Keyser and Witten, 2005) and that ecdysteroids regulate msslo mRNA levels in the DLMs, ecdysteroids could regulate the increases of msslo transcript levels in the CNS.…”

“…Late in adult development there is a dramatic and abrupt increase in msslo mRNA in the DLMs that temporally correlates to declining ecdysteroid titers (Kammer, 1979;Rheuben and Kammer, 1980;Bollenbacher et al, 1981;Keyser and Witten, 2005). This close temporal correlation suggests that the hormone mediates msslo mRNA levels.…”