Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle

Vogalis, Fivos; Vincent, Tonia L.; Qureshi, Ijaz A.; Schmalz, Felicitas; Ward, Manus W.; Sanders, Kenton M.; Horowitz, Burton

doi:10.1152/ajpgi.1996.271.4.g629

Cited by 35 publications

(55 citation statements)

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“…1F) document the shifts to the left in channel gating along the voltage axis with increasing free intracellular calcium. These observations are thus similar to those recently reported by others (33)(34)(35)(36)(37)(38)(39) for heterologous expression of mammalian Slo genes encoding BK Ca channels.…”

Section: Resultssupporting

confidence: 92%

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

Ling

Woronuk

et al. 2000

Journal of Biological Chemistry

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Large conductance, calcium-sensitive K ؉ channels (BK Ca channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK Ca channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK Ca channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage-and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK Ca channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK Ca channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK Ca channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK Ca channels underwent direct tyrosine phosphorylation in cells co-transfected with BK Ca channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK Ca channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.In the large family of voltage-gated K ϩ channels, large conductance, calcium-sensitive potassium (maxi-K or BK Ca ) 1 channels represent a unique class whose gating depends primarily on membrane voltage but which can be shifted in the negative direction by intracellular free calcium. A direct physiologic consequence of this behavior is that BK Ca channels act as "coincidence detectors" and regulate, in a feedback fashion, cellular processes stimulated by close temporal changes in membrane potential and intracellular calcium. That BK Ca channels indeed play such a role is evidenced by the fact that blocking these channels increases the degree of myogenic tone observed in arterial smooth muscle (1-3) and enhances the presynaptic calcium-dependent release of neurotransmitter at neuromuscular junctions (4, 5).Given their potential to influence cellular processes, it is not surprising that BK Ca channels are also targets of cellular signaling pathways, including phosphorylation and dephosphorylation reactions (6 -11), heterotrimeric GTP-binding proteins (12, 13), and the endothelium-derived vasodilator nitric oxide (14). To date, however, many of the molecular aspects of these regulatory events remain poorly understood.Of these various cellular pathways, protein phosphorylation remains as one of the most common forms of intrace...

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Section: Resultssupporting

confidence: 92%

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

Ling

Woronuk

et al. 2000

Journal of Biological Chemistry

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“…It is remarkable that cloned maxi-K channels seem not to respond to purified PKG after patch excision. PKG failed to activate Cslo (canine maxi-K channel) (28), which has an amino acid sequence almost identical to that of Hslo and has a conserved strong consensus site for PKG phosphorylation (27). In our experiments, purified PKG-I␣ failed to activate Hslo channels in 12 out of 13 inside-out patches, including patches pretreated with alkaline phosphatase (seven out of seven).…”

Section: Purified Pkg-i␣ Induces An Increase In the Open Probability mentioning

confidence: 53%

“…This possible PKG phosphorylation site is conserved in all mammalian maxi-K (slo) channels and is localized within its C-terminal region (27). However, purified PKG has failed to activate cloned maxi-K channels in excised inside-out membrane patches (28).…”

mentioning

confidence: 99%

The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

Alioua¹,

Tanaka²,

Wallner³

et al. 1998

Journal of Biological Chemistry

165

129

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Native large conductance, voltage-dependent, and Ca 2؉ -sensitive K ؉ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human poreforming ␣-subunit of the Ca 2؉ -sensitive K ؉ channel, Hslo, and the ␣-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ϳ20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates ␥-33 P into the immunopurified Hslo band of ϳ125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the ␣-subunit of large conductance Ca 2؉ -sensitive K ؉ channels is substrate for G-I␣ kinase in vivo and support direct phosphorylation as a mechanism for PKG-I␣-induced activation of maxi-K channels.

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“…Because CO increases the apparent Ca 2ϩ sensitivity of K Ca channels in newborn arteriole smooth muscle cells, we sought to determine whether this effect was dependent on the presence of a ␤-subunit. To address this question, we studied CO regulation of cslo-␣ subunits, which are expressed in arterial smooth muscle and can be activated via PKG-mediated phosphorylation (11,43). Cslo-␣ subunit DNA was transfected into HEK-293 cells, which do not express endogenous ␤-subunits (12).…”

Section: Co Activates K Ca Channels In Excised Membrane Patchesmentioning

confidence: 99%

Carbon monoxide activates K_Cachannels in newborn arteriole smooth muscle cells by increasing apparent Ca²⁺sensitivity of α-subunits

Tcheranova

Parfenova³

et al. 2004

American Journal of Physiology-Heart and Circulatory Physiology

104

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Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle

Cited by 35 publications

References 0 publications

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

Carbon monoxide activates K_Cachannels in newborn arteriole smooth muscle cells by increasing apparent Ca²⁺sensitivity of α-subunits

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Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle

Cited by 35 publications

References 0 publications

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

The Large Conductance, Voltage-dependent, and Calcium-sensitive K+ Channel, Hslo, Is a Target of cGMP-dependent Protein Kinase Phosphorylation in Vivo

Carbon monoxide activates KCachannels in newborn arteriole smooth muscle cells by increasing apparent Ca2+sensitivity of α-subunits

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Carbon monoxide activates K_Cachannels in newborn arteriole smooth muscle cells by increasing apparent Ca²⁺sensitivity of α-subunits