A Novel Current Pathway Parallel to the Central Pore in a Mutant Voltage-gated Potassium Channel

Prütting, Sylvia; Grissmer, Stephan

doi:10.1074/jbc.m110.185405

Cited by 9 publications

(35 citation statements)

References 49 publications

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“…As there is no K v 1.3 crystal structure, a template of the K v 1.3 S5/S6 domains was generated using the pdb-file of the crystal structure of the K v 1.2 channel (PDB: 2A79) with a sequence identity over 90% to the K v 1.3 channel. The K v 1.3 homology model was optimized following the instructions as described earlier [14]. All cysteine mutations were constructed by PyMOL (The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC).…”

Section: Methodsmentioning

confidence: 99%

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

Diesch

Grissmer²

2017

Cell Physiol Biochem

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Background/Aims: The human-voltage gated K_v1.3 channel (hK_v1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hK_v1.3 channels. We characterized the effect of verapamil on currents through hK_v1.3 channels paying special attention to the on-rate (k_on) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated. Methods: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hK_v1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate k_on of verapamil. Results: The on-rate of verapamil to block current through hK_v1.3_T419C mutant channels is similar to that obtained for hK_v1.3_wt channels whereas the on-rate of verapamil to block currents through hK_v1.3_L417C and hK_v1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hK_v1.3_L346C and the double mutant hK_v1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hK_v1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold. Conclusions: We conclude that position 420 in hK_v1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hK_v1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hK_v1.3_L346C and hK_v1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.

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Section: Methodsmentioning

confidence: 99%

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

Diesch

Grissmer²

2017

Cell Physiol Biochem

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“…Interestingly, this pathway had a permeability for cations that is comparable with the EAP described in this study (Na ϩ Ͼ NH 4 ϩ Ͼ Cs ϩ Ͼ K ϩ ). Furthermore, analogous to the EAP, current flow through this parallel current pathway was unaffected by pore blockers like charybdotoxin and tetraethylammonium (56). A comparable pathway running in parallel to the central pore was also proposed from molecular dynamics simulations in KcsA channels (57).…”

Section: Discussionmentioning

confidence: 84%

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

Lukács

Gawali

Cervenka

et al. 2014

Journal of Biological Chemistry

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“…at potentials where the α-pore is normally closed. The inward current of the mutant channel was similar but not identical to the ω-current in the VSD-mutant channels, for example, Na + and Li + could carry large inward σ-current whereas ions like K + and Rb + only generated small inward σ-currents [10, 11]. In addition, current through the σ-pore was blocked by verapamil and not influenced by CTX, KTX or MTX [10, 11].…”

Section: Introductionmentioning

confidence: 99%

“…The inward current of the mutant channel was similar but not identical to the ω-current in the VSD-mutant channels, for example, Na + and Li + could carry large inward σ-current whereas ions like K + and Rb + only generated small inward σ-currents [10, 11]. In addition, current through the σ-pore was blocked by verapamil and not influenced by CTX, KTX or MTX [10, 11]. According to the model of the mutant h Kv1.3_V388C channel [10], the σ-pore was located behind the central α-pore at the back of the selectivity filter and proceeded in parallel to the central α-pore.…”

Section: Introductionmentioning

confidence: 99%

“…A different ion conducting pathway, the σ-pore, was described in mutant voltage-gated potassium channels in which a valine was replaced by a cysteine at position 388 in h K v 1.3 channels (Shaker position 438) or at the homologue position 370 in h K v 1.2 channels [10, 11]. This mutation showed, in addition to the current through the central, potassium-selective α-pore, an additional inward current at membrane potentials more negative than -100 mV, i.e.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels

Tyutyaev¹,

Grissmer²

2018

Cell Physiol Biochem

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Background/Aims: The replacement of the amino acid valine at position 388 (Shaker position 438) in hK_v1.3 channels or at the homologue position 370 in hK_v1.2 channels resulted in a channel with two different ion conducting pathways: One pathway was the central, potassium-selective α-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hK_v1.3_V388C channel and CTX or MTX in the hK_v1.2_V370C channel). The other pathway (σ-pore) was behind the central α-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central α-pore was closed. In addition, current through the σ-pore could not be reduced by CTX, KTX or MTX in the hK_v1.3_V388C or the hK_v1.2_V370C channel, respectively. Methods: For a more detailed characterization of the σ-pore, we created a trimer consisting of three hK_v1.3_V388C α-subunits linked together and characterized current through this trimeric hK_v1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hK_v1.3_V388C channel by replacing single amino acids in the tetrameric hK_v1.3_V388C mutant channel that could be involved in σ-pore formation. Results: Overexpression of the trimeric hK_v1.3_V388C channel in COS-7 cells yielded typical σ-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hK_v1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channel were similar: currents could be observed at potentials more negative than -100 mV, were not carried by protons or chloride ions, and could not be reduced by peptide toxins (CTX, MTX) or TEA. The σ-pore was mostly permeable to Na⁺ and Li⁺. In addition, in our site-directed mutagenesis experiments, we created a number of new double mutant channels in the tetrameric hK_v1.3_V388C background channel. Two of these tetrameric double mutant channels (hK_v1.3_V388C_T392Y and hK_v1.3_V388C_Y395W) did not show currents through the σ-pore. Conclusions: From our experiments with the trimeric hK_v1.3_V388C channel we conclude that the σ-pore exists in hK_v1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hK_v1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

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A Novel Current Pathway Parallel to the Central Pore in a Mutant Voltage-gated Potassium Channel

Cited by 9 publications

References 49 publications

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels

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