Diverse Trafficking Patterns Due to Multiple Traffic Motifs in G Protein-Activated Inwardly Rectifying Potassium Channels from Brain and Heart

Ma, Dzwokai; Zerangue, Noa; Raab‐Graham, Kimberly F.; Fried, Sharon R.; Jan, Yuh Nung

doi:10.1016/s0896-6273(02)00614-1

Cited by 197 publications

(226 citation statements)

References 47 publications

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“…By taking the proposed arrangement of the P2X 2/3 subunits as valid, this suggests that one P2X 2 subunit is sufficient to stabilize the whole channel complex at the cell surface. Similar rescue of the surface expression of trafficking deficient ion channels by Wt subunits has been reported for the homo-oligomeric Kir2.1 and hetero-oligomeric Kir3 inward rectifier potassium channel (33). Kir channels have a tetrameric subunit organization; however, the number of Wt Kir subunits that are sufficient to drive the surface expression of the channel complex has not been documented.…”

Section: Discussionmentioning

confidence: 61%

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

Chaumont

Jiang

Penna

et al. 2004

Journal of Biological Chemistry

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Extracellular ATP-gated channels (P2X receptors) define the third major family of ionotropic receptors, and they are expressed widely in nerve cells, muscles, and endocrine and exocrine glands. P2X subunits have two membrane-spanning domains, and a receptor is thought to be formed by oligomerization of three subunits. We have identified a conserved motif in the cytoplasmic C termini of P2X subunits that is necessary for their surface expression; mutations in this motif result in a marked reduction of the receptors at the plasma membrane because of a rapid internalization. Transfer of the motif to a reporter protein (CD 4 ) enhances the surface expression of the chimera, indicating that this motif is likely involved in the stabilization of P2X receptor at the cell surface. In neurons, mutated P2X 2 subunits showed reduced membrane expression and an altered axodendritic distribution. This motif is also present in intracellular regions of other membrane proteins, such as in the third intracellular loop of some G protein-coupled receptors, suggesting that it might be involve in their cellular stabilization and polarization.P2X receptors are extracellular ATP-gated ion channels; they define the third major class of ligand-gated channels together with the glutamate and the nicotinic superfamilies (1). P2X receptors are involved in synaptic transmission in the central and peripheral nervous systems, but in contrast to other ligand-gated channels, they are also widely expressed in peripheral tissues where they participate in physiological processes as diverse as smooth muscle contraction, secretion, and bone resorption. ATP-induced currents have been recorded from a large number of different cell types (2), but the molecular identity of P2X receptors responsible for these currents often remains elusive because of limitations of pharmacological tools to discriminate the different subtypes of P2X channels.Seven P2X subunit genes (P2X 1 -P2X 7 ) are found in the human genome; this number seems to be an accurate estimation of the extent of the P2X family in mammalian species, although in zebrafish two additional subunits appear to exist (2, 3). The basic structural determinants of P2X channels have been established by numerous molecular studies (4). P2X subunits have a membrane topology with two transmembrane domains linked by a large extracellular loop; N and C termini are localized intracellularly (5, 6). To form a channel, P2X subunits are thought to associate as homo-or heterooligomers, composed of three subunits (7, 8) organized in a head-to-tail orientation around a central pore (9). This model is consistent with studies that show that the permeation pathway is associated with transmembrane regions (10 -12), and with the fact that the gating of the channel is in part due to movements of the subunits relative to the each other (12). Conserved charged residues in the extracellular loop have been implicated in ATP binding (13,14), and desensitization is tightly linked to transmembrane domains and intracellular regions lo...

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Section: Discussionmentioning

confidence: 61%

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

Chaumont

Jiang

Penna

et al. 2004

Journal of Biological Chemistry

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“…Previous studies have shown that an epitope insertion in these positions has no detectable effects on the function of Kir channels or GPCRs (19)(20)(21)(22). Compared with an empty vector control, overexpression of AGS3 caused an increase in the total protein levels of most receptors and channels assayed (Fig.…”

mentioning

confidence: 79%

A specific role of AGS3 in the surface expression of plasma membrane proteins

Groves

Gong

et al. 2007

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

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Activator of G protein signaling 3 (AGS3), originally identified in a functional screen for mammalian proteins that activate heterotrimeric G protein signaling, is known to be involved in drug-seeking behavior and is up-regulated during cocaine withdrawal in animal models. These observations indicate a potential role for AGS3 in the formation or maintenance of neural plasticity. We have found that the overexpression of AGS3 alters the surface-to-total ratios of a subset of heterologously expressed plasma membrane receptors and channels. Further analysis of the endocytic trafficking of one such protein by a biotin-based internalization assay suggests that overexpression of AGS3 moderately affects the internalization or recycling of surface proteins. Moreover, AGS3 overexpression and siRNA-mediated knockdown of AGS3 both result in the dispersal of two endogenously expressed trans-Golgi network (TGN)-associated cargo proteins without influencing those in the cis-or medial-Golgi compartments. Finally, adding a TGN-localization signal to a CD4-derived reporter renders the trafficking of fusion protein sensitive to AGS3. Taken together, our data support a model wherein AGS3 modulates the protein trafficking along the TGN/plasma membrane/endosome loop. drug addiction ͉ Golgi apparatus ͉ membrane trafficking ͉ receptors and channels

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“…Thus, we determined whether this co-assembly with L18A/L25A would inhibit cell surface expression of wild type channel. For these studies we transiently co-transfected wild type and mutated channel into HEK cells and evaluated cell surface expression of the HA-tagged channel using both IF as well as a chemiluminescence assay (29). As shown in Fig.…”

Section: Fig 2 Mutations In the Nh 2 -Terminal Dileucine And Leucinmentioning

confidence: 99%

“…6A, right panel). These effects were more quantitatively addressed using the chemiluminescence-based assay devised by Jan and colleagues (29). As shown in Fig.…”

Section: Fig 2 Mutations In the Nh 2 -Terminal Dileucine And Leucinmentioning

confidence: 99%

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

Jones

Hamilton

Papworth

et al. 2004

Journal of Biological Chemistry

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The role of the NH 2 -terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH 2 -terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and wholecell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 ؎ 1.3 min compared with 3.2 ؎ 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH 2 terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27°C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH 2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.The KCNN gene family is composed of four members, including the small conductance Ca 2ϩ -activated K ϩ channels (SK1, SK2, and SK3) 1 and the intermediate conductance Ca 2ϩ -activated K ϩ channel (IK1 or SK4). While the IK1 and SK channels share roughly 40% homology, their expression patterns and pharmacology are widely disparate, consistent with their unique physiological functions (1). The human IK1 channel (hIK1) is widely expressed, being found in salivary gland, colon, bladder, stomach, lung, smooth muscle, red blood cells, T-cells, and placenta (2, 3) where it plays a crucial role in a variety of physiological functions. Indeed hIK1 plays a seminal role in modulating Ca 2ϩ -dependent transepithelial ion transport (4, 5), has recently been confirmed to be the Gardos channel of red blood cells (6), and has been proposed to play a role in both vascular remodeling (7) and in modulating vascular tone (8). Because of these critical physiological roles, the ...

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Diverse Trafficking Patterns Due to Multiple Traffic Motifs in G Protein-Activated Inwardly Rectifying Potassium Channels from Brain and Heart

Cited by 197 publications

References 47 publications

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors

A specific role of AGS3 in the surface expression of plasma membrane proteins

Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1

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