Ca<sup>2+</sup> activation of hSlo K<sup>+</sup> channel is suppressed by N‐terminal GFP tag

Meyer, Elisabeth; Fromherz, Peter

doi:10.1046/j.1460-9568.1999.00548.x

Cited by 22 publications

(22 citation statements)

References 14 publications

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“…Bravo-Zehnder et al (45) also reported that myc-tagged MaxiK channel has characteristics similar to those of wild type. N terminus GFP tagging of hslo changes the channel's electrophysiological properties (46). In our case, the GFP-tagged rbslo2 did not generate currents, like the wild type, and its intracellular distribution was not changed.…”

Section: Discussionmentioning

confidence: 52%

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

Wang

Ikeda

Guggino

2003

Journal of Biological Chemistry

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The large conductance, voltage-and Ca 2؉ -activated K ؉ channel (MaxiK) is expressed in several renal segments and functions in cell volume regulation and flowmediated K ؉ secretion. Previously, we cloned two MaxiK channel isoforms, named rbslo1 and rbslo2, from rabbit renal cells. rbslo1 has a 58-amino acid insertion after the S8 hydrophobic domain, whereas rbslo2 is truncated and cannot be activated. Here we use the sequence differences between the two variants to examine their plasma membrane processing. Plasma membrane localization of rbslo1 and 2 expressed in HEK293 cells was assayed by electrophysiology, immunocytochemistry, and biochemistry studies. Consistent with its functional silence, rbslo2 localized primarily within the cytoplasm, presumably in the endoplasmic reticulum and Golgi region. Coexpression with MaxiK ␤ subunits did not alter the cellular localization of either rbslo1 or rbslo2. When rbslo1 and 2 are cotransfected in non-polarized cells, they colocalized primarily within the cell with only rbslo1 detected at the plasma membrane. When transfected into polarized, medullary-thick ascending limb (mTAL) cells, rbslo1 is expressed at the apical membrane whereas the majority of rbslo2 localized throughout the cytoplasm. Given the high degree of similarity between the two isoforms, we conclude that the cytoplasmic tail of rbslo1 is important for the cell surface expression of MaxiK channels.

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Section: Discussionmentioning

confidence: 52%

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

Wang

Ikeda

Guggino

2003

Journal of Biological Chemistry

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“…In particular, Khakh and colleagues (15) revealed a right- ward shift in the ATP dose-response curve of the P2X2-GFP construct when expressed in Xenopus oocytes, although there was no change in the Hill coefficient. GFP fused to the amino terminus altered the kinetics of K ϩ channels, although GFP addition to the carboxy terminus was not examined (18,23). The present study highlights the importance of functional testing of both amino-and carboxy-terminal fusion constructs before other applications.…”

Section: Discussionmentioning

confidence: 85%

Pore formation is not associated with macroscopic redistribution of P2X7 receptors

Smart

Panchal

Bowser

et al. 2002

American Journal of Physiology-Cell Physiology

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The present study examines whether changes in P2X7 purinergic receptor density precede formation of the cytolytic pore characteristic of this receptor. We fused P2X7 receptors with enhanced green fluorescent protein (EGFP) at the amino or carboxy termini (EGFP-P2X7 and P2X7-EGFP). Electrophysiological characterization in Xenopus oocytes revealed wild-type responses to ATP for GFP-tagged receptors. However, differences in sensitivity to ATP were apparent with the P2X7-EGFP receptor displaying a threefold reduction in ATP sensitivity compared with control. Ethidium ion uptake was used to measure cytolytic pore formation. Comparison of tagged receptors with wild type in HEK-293 and COS-7 cells showed there was no significant difference in ethidium ion uptake, suggesting that fusions with EGFP did not interfere with cytolytic pore formation. Confocal microscopy confirmed that tagged receptors localized to the plasmalemma. Simultaneous monitoring of EGFP and ethidium ion fluorescence revealed that changes in receptor distribution do not precede pore formation. We conclude that it is unlikely that large scale changes in P2X7 receptor density precede pore formation.

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“…Such a positive modulation of the level of expression has not been described for neurotransmitter receptors tagged with GFP. Placing the GFP in the extracellular amino terminus of the hSlo Ca 2ϩ -activated K ϩ channel reduced its activation by affecting both the voltage and Ca 2ϩ dependence of activation (14). In the case of GABA1, tagging GFP to the extracellular carboxyl terminus led to slower incorporation of receptors into the plasma membrane and reduced the amplitude of GABA-currents (18).…”

Section: Discussionmentioning

confidence: 99%

“…Despite the increasing use of fluorescent tagging to visualize receptors and other cellular proteins, there is no report on the functional properties of GFP-tagged AMPA receptors. Whereas GFP tagging does not seem to alter the function of some receptors (10)(11)(12)(13), it is known that for some receptors GFP alters their function (14)(15)(16), membrane localization (17) or rate of membrane incorporation (18). With this question in mind, we analyzed the properties of homomeric GFP-tagged GluR3 receptors expressed in Xenopus oocytes.…”

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confidence: 99%

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

Limón

Reyes-Ruiz

Eusebi

et al. 2007

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

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Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC 50 values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate-to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo-[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins.fluorescent tag ͉ receptor expression ͉ Xenopus oocytes ͉ kainate ͉ glutamate G lutamate is the main excitatory neurotransmitter in the mammalian central nervous system. Its receptors can either link to a second messenger receptor channel-coupling system (metabotropic) or can themselves form ligand-gated ion channels (ionotropic) (1, 2). The ionotropic glutamate receptors are divided into three groups, according to their differential affinity for the agonists N-methyl-D-aspartate (NMDA), kainate (Kai), and ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (2). Functional AMPA receptors are made up of homomeric or heteromeric arrangements of the glutamate receptor (GluR)-1 to -4 subunits; their membrane topology indicates an extracellular amino terminus, three transmembrane segments, one membrane reentrant loop, and an intracellular carboxyl-terminal end (3, 4).

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Ca²⁺ activation of hSlo K⁺ channel is suppressed by N‐terminal GFP tag

Cited by 22 publications

References 14 publications

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

Pore formation is not associated with macroscopic redistribution of P2X7 receptors

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

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Ca2+ activation of hSlo K+ channel is suppressed by N‐terminal GFP tag

Cited by 22 publications

References 14 publications

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

The Cytoplasmic Tail of Large Conductance, Voltage- and Ca2+-activated K+ (MaxiK) Channel Is Necessary for Its Cell Surface Expression

Pore formation is not associated with macroscopic redistribution of P2X7 receptors

Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

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Ca²⁺ activation of hSlo K⁺ channel is suppressed by N‐terminal GFP tag