K+ pore structure revealed by reporter cysteines at inner and outer surfaces

Pascual, Juan M.; Shieh, Char‐Chang; Kirsch, Glenn E.; Brown, Arthur M.

doi:10.1016/0896-6273(95)90344-5

Cited by 107 publications

(105 citation statements)

References 31 publications

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“…Based on the substituted cysteine accessibility method (10), as well as internal tetraethylammonium blockade (7), central residues have been predicted to face the cytoplasmic side and thus penetrate the membrane. On the other hand, a Kv channel, Herg, has a utilized native N-glycosylation sequon that directly follows the invariant G(Y/F)G sequence (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Schwalbe

Rudin

Xia

et al. 2002

Journal of Biological Chemistry

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Inwardly rectifying K؉ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K ؉ channels, Kvs, and this stretch is called the signature sequence of K ؉ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K ؉ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation

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Section: Discussionmentioning

confidence: 99%

“…Voltage-dependent K ϩ channels, Kvs, have a highly homologous H5 segment that appears to be the major pore determinant (see Fig. 1B) (7)(8)(9)(10). H5 contains eight highly conserved residues and has been proposed as the selectivity filter of K ϩ channels (see Fig.…”

mentioning

confidence: 99%

Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Schwalbe

Rudin

Xia

et al. 2002

Journal of Biological Chemistry

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“…selectivity filter were either non-functional or insensitive to external MTS modification (42) just as we have shown in ECaC-TRPV5. The alignment produced by PROFILES-3D would correlate A545C in ECaC-TRPV5 with Y380C in Kv2.1 (42). These residues were shown to be functionally modified by MTSEA, MTSET, and MTSES in both channels.…”

Section: Fig 3 the Endogenous Cysteine At Position 556 Accounts Formentioning

confidence: 99%

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

Dodier

Banderali

Klein

et al. 2004

Journal of Biological Chemistry

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The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu 515 ) to the beginning of S6 (Ala 560 ). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro 527 and Ile 541 was compatible with the presence of a ␣-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions ؊1, 0, ؉1, ؉2 The TRP ion channels form a large class of cationic channels that are related to the product of the Drosophila TRP gene. TRP channels share a similar predicted topology of six transmembrane segments in which the amino acids that link the fifth and sixth transmembrane domains line the pore region (1). According to the recent IUPHAR classification of ion channels (2), the 21 members of the TRP family can be divided by sequence homology into three subfamilies (3, 4) as short (TRPCx), long or melastatin (TRPMx), and osm-9-like or vanilloid-like (TRPVx) channels. The molecular domains that are mostly conserved among TRP channels include part of the S6 segment, ankyrin repeats in the N terminus, and a "TRP domain" in the C terminus (EWKFAR) (5), the latter being absent from TRPV channels. The TRPC and TRPV proteins have 2-4 N-terminal ankyrin domains suggesting that these proteins are coupled to the spectrin-based membrane cytoskeleton.TRP channels vary significantly in their biophysical properties and gating mechanisms. In contrast to other members of the TRP family, TRPV5 and TRPV6 channels show strong inward rectification, exhibit anomalous mole-fraction effect, are activated by low [Ca 2ϩ ] i and inactivated by higher [Ca 2ϩ ] i (6 -8). TRPV5 and TRPV6 are also highly Ca 2ϩ -selective channels with PCa/PNa Ͼ 100. In particular, ECaC-TRPV5 displays a high Ca 2ϩ affinity with a K d of Ϸ2 M (7) that is comparable to the K d of Ϸ1 M for voltage-dependent Ca V channels (9). A single residue in the S5-S6 linker (Asp 542 ) was found to account for the high Ca 2ϩ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of a lysine residue, the Ϸ20-fold lower Ca 2ϩ selectivity of TRPV1-4 channels (10). TRPV5 and TRPV6 channels can also form homo-and heterotetramers suggesting that they are structurally and functionally related (11).There is currently very little structural data available on the pore architecture of Ca 2ϩ -selective TRP channels. It...

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“…Some of this work has focused on site-specific mutagenesis of amino acids in the P-region, a highly conserved loop of 21 amino acids present in all cloned K channels (e.g., Yool and Schwarz, 1991;Kirsch et al, 1992;Heginbotham et al, 1994;Kirschet al, 1995). Other efforts have been directed toward experimentally determining aspects of the physical structure of this region (K/irz et al, 1995;Lfi and Miller, 1995;Pascual et al, 1995) or modeling pore structure (Durrell and Guy, 1992;Durrell, 1994,1995;Mackinnon, 1995).…”

Section: Introductionmentioning

confidence: 99%

Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes.

Stampe

Begenisich

1996

The Journal of General Physiology

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A B S T R A C T We describe a method to evaluate the ratio of ionic fluxes through recombinant channels expressed in a single Xenopus oocyte. A potassium channel encoded by the Drosophila Shakergene tested by this method exhibited flux ratios far from those expected for independent ion movement. At a fixed extracellular concentration of 25 mM K +, this channel showed single-file diffusion with an Ussing flux-ratio exponent, n', of 3.4 at a membrane potential of -30 mV. There was an apparent, small voltage dependence of this parameter with n' values of 2.4 at -15 and -5 inV. These results indicate that the pore in these channels can simultaneously accommodate at least four K + ions. If each of these K + ions is in contact with two water molecules, the minimum length of the pore is 24 A.

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K+ pore structure revealed by reporter cysteines at inner and outer surfaces

Cited by 107 publications

References 31 publications

Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Site-directed Glycosylation Tagging of Functional Kir2.1 Reveals That the Putative Pore-forming Segment Is Extracellular

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes.

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