Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line

Klaiber, Kimberly; Williams, Nidhi Gupta; Roberts, Thomas M.; Papazian, Diane M.; Jan, Yuh Nung; Miller, Christopher

doi:10.1016/0896-6273(90)90311-3

Cited by 94 publications

(70 citation statements)

References 28 publications

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“…Similar observations were also made for animal ion channels, e.g. the activation curve of Shaker K + channels expressed in Sf9 cells shifted towards more depolarized potentials with respect to its expression in oocytes [15]. Thus the discrepancy in permeability and voltage-dependent gating of KAT1 in the insect cell line is probably related to the expression system.…”

Section: Resultssupporting

confidence: 75%

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Functional expression of the plant K⁺ channel KAT1 in insect cells

et al. 1996

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Section: Resultssupporting

confidence: 75%

“…Sf9 cells have been reported as a suitable system for the functional expression and purification of animal ion channels [2,5,15,16]. But to our knowledge, insect cells were not yet used to express and to study plant ion channels.…”

Section: Introductionmentioning

confidence: 99%

Functional expression of the plant K⁺ channel KAT1 in insect cells

et al. 1996

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“…Cells were grown in monolayer at 260C. These experiments employed a recombinant virus, Shaker 3A1, containing cDNA derived from the Drosophila Shaker H4 (10) "A-type" K+ channel placed directly behind the polyhedrin promoter (11). Shaker currents were assayed 1 or 2 days after infection of Sf9 cells with this recombinant virus, exactly as described (11).…”

Section: Methodsmentioning

confidence: 99%

Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

Armstrong

Miller

1990

Proc. Natl. Acad. Sci. U.S.A.

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Removal of Ca2+ from the solution bathing neurons is known in many cases to alter the gating properties of voltage-dependent K+ channels and to induce a large, nonselective "leak" conductance. We used a heterologous expression system to test whether the leak conductance observed in neurons is mediated by voltage-dependent K+ channels in an altered, debased conformation. Voltage-dependent K+ channels were expressed in an insect cell line infected with a recombinant baculovirus carrying the cDNA for Drosophila Shaker "A-type" K+ channels. These expressed channels respond to low Ca21 identically to voltage-dependent K+ channels in native neuronal membranes; upon removal of external Ca2+, Shaker K+ currents disappear and are replaced by a steady, nonselective leak conductance. However, control cells devoid of Shaker channels were free of any voltagedependent conductances and did not generate a leak when external Ca2+ was removed. These results show that Ca2+ is essential for proper function of voltage-dependent K+ channels and is required to stabilize the native conformations of these membrane proteins.needed to maintain the native structure of voltage-dependent K+ channels. According to this picture, Ca2+ removal partially denatures the channel so that its pore loses the ability to discriminate among small ions and its voltage-dependent "gate" is unable to close.One problem that makes this interpretation equivocal is that the neuronal membrane is electrically complicated, providing a home for many different types of ion channels. The experiments in neurons do not allow us to conclude with certainty that the leak induced by Ca2+ removal is in fact mediated by the specific set of voltage-dependent K+ channels under study, rather than by other membrane proteins present. The experiments here are designed to test this idea rigorously; we examine the effect of Ca2+ removal in a non-neuronal cell line in which a heterologous voltagedependent K+ channel is specifically expressed by infection with a recombinant baculovirus. The results clearly demonstrate that the nonselective leak induced by removing Ca2+ has its origin in a massive functional alteration of the expressed K+ channels.Voltage-dependent K+ channels play a central role in the generation, propagation, and integration of electrical signals in many types of cells. These channels all display high selectivity for K+ over Na+ and strong voltage dependence of opening and closing rates. The long-recognized functional similarities among voltage-dependent K+ channels are now being rationalized in terms of homologies among members of a very large molecular family (1-5). Because membrane voltage is the physiological signal used to switch these channels between open and closed conformations, relatively little attention has been given to the roles of physiological ligands in their proper functioning. In particular, Ca2+, which is crucially involved in the action of Ca2+-activated K+ channels and many membrane-transducing systems, is not generally viewed as being dir...

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“…To study the mechanism of protein-mediated membrane fusion, it would be useful to hay0 a simple expression system for a fusion protein which would allow for optical and electrophysiological studies of fusion pore formation, as well as permit genetic alteration of fusion protein sequence. The baculovirus/insect cell system [1 ] has been used to advantage in electrophysiological studies of recombinant channels [2]. Influenza hemagglutinin [3], VSV G protein [4], and the fusion proteins from measles [5], bovine coronavirus [6], and human immunodeficiency virus [7] have all been successfull), expressed in Spodoptera frugiperda (Sf9) insect cells using baculovirus, with fusion activity demonstrated as pH-dependent syncytia formation [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Acidic pH induces fusion of cells infected with baculovirus to form syncytia

1992

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The enveloped baculoviras\insect cell system has been used extensively for expression of recombinant proteins, including viral fusion proteins. We tested wiid-tyl~ baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance chanses were observed after incubating infected Spodopterafrugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse of 10 s is needed to trisect syncytia formation. Identical results were obtained with recombinant baculovirus. This new ~ys:e,r~ !,_ convenient for studying pH activated ccll~dl fusion. However, using this enveloped virus to study ~he mechanism of recomlHzant fusion proteins rexluires caution.

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Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line

Cited by 94 publications

References 28 publications

Functional expression of the plant K⁺ channel KAT1 in insect cells

Functional expression of the plant K⁺ channel KAT1 in insect cells

Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

Acidic pH induces fusion of cells infected with baculovirus to form syncytia

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Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line

Cited by 94 publications

References 28 publications

Functional expression of the plant K+ channel KAT1 in insect cells

Functional expression of the plant K+ channel KAT1 in insect cells

Do voltage-dependent K+ channels require Ca2+? A critical test employing a heterologous expression system.

Acidic pH induces fusion of cells infected with baculovirus to form syncytia

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Functional expression of the plant K⁺ channel KAT1 in insect cells

Functional expression of the plant K⁺ channel KAT1 in insect cells