The Roles of N- and C-terminal Determinants in the Activation of the Kv2.1 Potassium Channel

Ju, Ming‐Shaung; Stevens, Louisa; Leadbitter, E; Wray, Dennis

doi:10.1074/jbc.m212973200

Cited by 62 publications

(78 citation statements)

References 28 publications

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“…Schulteis et al (31) showed that exposure of cells expressing Kv1.1 to oxidizing conditions causes the formation of a disulfide bond between a cysteine in the N-terminal T1 domain and a cysteine in the C terminus of the adjacent subunit. Also, an interaction between the N terminus and the C terminus of Kv2.1 is responsible for the gating process (32)(33)(34). Furthermore, an interaction between the N terminus and the C terminus of TRPV5 has been identified by a GST pull-down assay and coimmunoprecipitation (28).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, the functional results obtained with the chimeras [N-IVT4]T6, [N-VIT4]T6, and [CT4]T6 suggest that the interaction between the N terminus and the C terminus is not absolutely required to assemble a functional TRPC channel. However, this interaction may be involved in the regulation of the activity of the channel, as is the case for Kv2.1 (32). Another possibility is that different assembly phases must occur to get the functional conformation of the channel.…”

Section: Discussionmentioning

confidence: 99%

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Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Lepage

Lussier

Barajas-Martínez

et al. 2006

Journal of Biological Chemistry

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Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo-or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G q proteincoupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.Calcium signaling is involved in many cellular functions, including cell growth, differentiation, contraction, and secretion. The elevation of [Ca 2ϩ ] i occurs via two distinct phases upon activation of a G q protein-coupled receptor or a tyrosine kinase receptor. The first phase is mediated through the activation of phospholipase C, which catalyzes the production of inositol 1,4,5-trisphosphate, which in turn mobilizes Ca 2ϩ from the endoplasmic reticulum. The second phase of [Ca 2ϩ ] i elevation involves Ca 2ϩ entry from the extracellular milieu that maintains the [Ca 2ϩ ] i higher than the basal level (1, 2). The transient receptor potential canonical (TRPC) 3 protein family is involved in calcium entry and is composed of seven members (TRPC1 to TRPC7) that are calcium-permeable cation channels (3, 4). Based on sequence similarity, TRPCs can be further subdivided into four groups. Groups 1 and 2 contain exclusively the isoforms TRPC1 and TRPC2, respectively. Group 3 includes TRPC3, TRPC6, and TRPC7, whereas group 4 includes TRPC4 and TRPC5. Group 4 TRPCs are most closely related to group 1. TRPCs consist of a transmembrane domain composed of six segments with a putative pore between the fifth and the sixth transmembrane segments. The transmembrane domain is flanked with cytoplasmic N and C termini (5). TRPCs posse...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Lepage

Lussier

Barajas-Martínez

et al. 2006

Journal of Biological Chemistry

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“…They also participate in channel assembly in Kv 2.1 (13,14). All members of the Shaker subfamily of Kv channels display a remarkable amino acid sequence conservation within the distal region of their C termini, characterized by the -T͞SDV motif (10,12).…”

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confidence: 99%

“…Furthermore, Ju et al (14) have recently proposed that the gating of the Kv2.1 channel is strongly influenced by specific interactions between the N-and Cterminal domains, suggesting they do assume a well defined 3D structure. Finally, the 3D structure of the full-length Shaker potassium channel ␣-subunit at 25-Å resolution (17) revealed a larger membrane-embedded domain and a smaller cytoplasmic domain, conjoined by four thin connectors resembling a ''hanging gondola'' (18).…”

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confidence: 99%

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Sokolova

Accardi

Gutiérrez

et al. 2003

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

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We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (⌬C) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and ⌬C Shaker, assembled with the rat Kv␤2-subunit. By using a difference map between the full-length and truncated Shaker ␣-␤ complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the ␤-subunit. This conformational change, induced by the binding of the Kv␤2-subunit, suggests a possible mechanism for the modulation of the K ؉ voltage-gated channel function by its ␤-subunit.C-terminal deletion ͉ electron microscopy ͉ single-particle analysis

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“…Amino acids 67 and 75 of the N-terminal T1 domain and amino acids 740 -853 of the C-terminal region ("CTA" domain) have been shown to be involved in determining the time course of activation of the channel (19), and interactions between the T1 domain and the CTA domain have been demonstrated. We report here that relative voltage-gated rearrangements in the cytoplasmic domain of the K v 2.1 channel may be successfully probed by FRET between ECFP and EYFP fluorophores fused to the termini of the K v 2.1 monomers.…”

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confidence: 99%

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

Kobrinsky

Stevens

Kazmi

et al. 2006

Journal of Biological Chemistry

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The voltage-gated K v 2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltageinduced relative molecular rearrangements of the channel, K v 2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the K v 2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N-and C-terminal tags fused to the same or different K v 2.1 monomers decreased upon activation of the channel by depolarization from ؊80 to ؉60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the K v 2.1 N-or C-terminal tags and the membrane-trapped EYFP N -PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the K v 2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the K v 2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged K v 2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.Voltage-activated K ϩ channels are a large and diverse family of homotetrameric membrane proteins that are similar in their ability to select for K ϩ over other ions but show differences in kinetics and voltage dependence of activation and inactivation (1). Each of the K v ␣ subunits is assembled into six transmembrane domains that encode determinants of voltage sensing and ion selectivity. The N and C termini are located in the cytoplasm and support a number of important interactions. The N-terminal tetramerization (T1) domain determines the specificity of assembly of K v ␣ subunits (2-6). The crystal structure of the T1 domain is known and forms a tetramer below the membrane like a "hanging gondola" (7), while the C-terminal domain surrounds this N-terminal structure (8). There are voltage-dependent interactions between the N terminus and the C terminus that are important determinants of channel activation and modulate the activation time course of K v ␣ channels (6, 9).There was remarkable progress in investigation of molecular rearrangements of the membrane-spanning core K v ␣ protein associated with voltage gating of K ϩ channels (10 -12). A recent model (13) provided insight into the folding of the N terminus fragment of K v 1.2 and suggested potentially important association between the gating and C terminus. However, the size and direction of rearrangements that may occur upon gating ...

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The Roles of N- and C-terminal Determinants in the Activation of the Kv2.1 Potassium Channel

Cited by 62 publications

References 28 publications

Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

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The Roles of N- and C-terminal Determinants in the Activation of the Kv2.1 Potassium Channel

Cited by 62 publications

References 28 publications

Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Identification of Two Domains Involved in the Assembly of Transient Receptor Potential Canonical Channels

Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvβ2-subunit

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

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Conformational changes in the C terminus of Shaker K ⁺ channel bound to the rat Kvβ2-subunit