Structure Acquisition of the T1 Domain of Kv1.3 during Biogenesis

Kosolapov, Andrey; Tu, Li‐Wei; Wang, Jing; Deutsch, Carol

doi:10.1016/j.neuron.2004.09.011

Cited by 54 publications

(66 citation statements)

References 40 publications

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“…Cysteine 73C is located inside the ribosomal tunnel, whereas cysteine 72C is located outside the tunnel. 1,33 We treated the nascent peptide first with cysteine-modifying reagent (MTS or MAL) for various times, quenched with reducing agent, and then assessed modification using a pegylation assay of the residual free thiol. 31 A cysteine that has not been modified by MTS or MAL reagent during the allotted incubation time will be labeled subsequently with PEG-MAL, a mass-tag used to identify available cysteine sidechains.…”

Section: Resultsmentioning

confidence: 99%

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Kobertz

Deutsch

2007

Journal of Molecular Biology

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109

124

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Section: Resultsmentioning

confidence: 99%

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Kobertz

Deutsch

2007

Journal of Molecular Biology

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109

124

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“…Similarly, Western blot analysis of ENaC in COS-7 cells and precipitation of the 2 (AP50)-subunit of the AP-2 complex with ENaC followed standard methods (32,33,(37)(38)(39). In brief, COS-7 cells were transfected with Myc-tagged ␤-and ␥-and Myc, His 6 -tagged ␣-ENaC.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Epithelial Na+ Channel Activity by Conserved Serine/Threonine Switches within Sorting Signals

Staruschenko

Pochynyuk

Stockand

2005

Journal of Biological Chemistry

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The PY and YXX motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the 2 -subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na ؉ channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)). Just preceding the PY and embedded within the YXX motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr 628 preceding the PY motif in ␥-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of ␥-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of ␥-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in ␥-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr 628 and Thr 635 also disrupted ENaC precipitation with the 2 -subunit of the AP-2 complex. Moreover, the YXX motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXX motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.The epithelial Na ϩ channel (ENaC) 2 is localized to the luminal plasma membrane of epithelial cells, particularly those involved in fluid absorption and epithelia surface hydration (1-3). As a consequence of this location and its function, ENaC activity is often limiting for Na ϩ absorption with corresponding effects on fluid movement. The critical role played by ENaC in terrestrial vertebrates has become apparent through investigation of diseases resulting from loss and gain of function mutations in this ion channel and its upstream regulators. Loss of function mutations result in inappropriate renal salt wasting in humans associated with electrolyte imbalance and decreased blood pressure (4 -6). Moreover, in mice and possibly humans, loss of ENaC function also impairs fluid clearance of the fetal lung (7,8). Gain of function mutations in ENaC and its upstream regulators, in contrast, result in inappropriate renal salt conservation associated with hypertension (2, 9). Recent evidence, moreover, suggests that gain of ENaC function may also lead to dry air spaces similar to that in cystic fibrosis (10).ENaC is a heteromeric channel comprised of three similar but disti...

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“…The T1 domains in the N termini of Kv channels are conserved and they form tetramers (Li et al, 1992;Choe, 2002;Nanao et al, 2003;Long et al, 2005) right after emerging from the ribosomal exiting tunnel once synthesis of the T1-S1 linker is completed (Kosolapov et al, 2004). It is important to note that although the T1 domains are highly conserved within the Kv3 family (Ͼ 70% identify), the Kv3 T1 domain shares only ϳ40% homology with the Kv1 T1 domain and requires four Zn 2ϩ ions to assemble into tetramers (Nanao et al, 2003).…”

Section: Direct Interaction Between the T1 Domain And The Atm Regionmentioning

confidence: 99%

The Axon–Dendrite Targeting of Kv3 (Shaw) Channels Is Determined by a Targeting Motif That Associates with the T1 Domain and Ankyrin G

Xu¹,

Cao²,

Xiao³

et al. 2007

J. Neurosci.

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Kv3 (Shaw) channels regulate rapid spiking, transmitter release and dendritic integration of many central neurons. Crucial to functional diversity are the complex targeting patterns of channel proteins. However, the targeting mechanisms are not known. Here we report that the axon-dendrite targeting of Kv3.1 is controlled by a conditional interaction of a C-terminal axonal targeting motif (ATM) with the N-terminal T1 domain and adaptor protein ankyrin G. In cultured hippocampal neurons, although the two splice variants of Kv3.1, Kv3.1a and Kv3.1b, are differentially targeted to the somatodendritic and axonal membrane, respectively, the lysine-rich ATM is surprisingly common for both splice variants. The ATM not only directly binds to the T1 domain in a Zn 2ϩ -dependent manner, but also associates with the ankyrin-repeat domain of ankyrin G. However, the full-length channel proteins of Kv3.1b display stronger association to ankyrin G than those of Kv3.1a, suggesting that the unique splice domain at Kv3.1b C terminus influences ATM binding to T1 and ankyrin G. Because ankyrin G mainly resides at the axon initial segment, we propose that it may function as a barrier for axon-dendrite targeting of Kv3.1 channels. In support of this idea, disrupting ankyrin G function either by over-expressing a dominant-negative mutant or by siRNA knockdown decreases polarized axon-dendrite targeting of both Kv3.1a and Kv3.1b. We conclude that the conditional ATM masked by the T1 domain in Kv3.1a is exposed by the splice domain in Kv3.1b, and is subsequently recognized by ankyrin G to target Kv3.1b into the axon.

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Structure Acquisition of the T1 Domain of Kv1.3 during Biogenesis

Cited by 54 publications

References 40 publications

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Mapping the Electrostatic Potential within the Ribosomal Exit Tunnel

Regulation of Epithelial Na+ Channel Activity by Conserved Serine/Threonine Switches within Sorting Signals

The Axon–Dendrite Targeting of Kv3 (Shaw) Channels Is Determined by a Targeting Motif That Associates with the T1 Domain and Ankyrin G

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