PDBsum entry 1j95

Membrane protein

PDB id: 1j95

Contents

Protein chains

98 a.a. *

Ligands

TBA

Metals

__K ×4

* Residue conservation analysis

PDB id:

1j95

Name:

Membrane protein

Title:

Kcsa potassium channel with tba (tetrabutylammonium) and potassium

Structure:

Voltage-gated potassium channel. Chain: a, b, c, d. Fragment: residues 1-125. Engineered: yes. Mutation: yes

Source:

Streptomyces lividans. Organism_taxid: 1916. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Octamer (from PQS)

Resolution:

2.80Å R-factor: 0.298 R-free: 0.322

Authors:

J.H.Morais-Cabral,R.Mackinnon,M.Zhou

Key ref:

M.Zhou et al. (2001). Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature, 411, 657-661. PubMed id: 11395760 DOI: 10.1038/35079500

Date:

23-May-01 Release date: 13-Jun-01

Protein chains

P0A334  (KCSA_STRLI) -  pH-gated potassium channel KcsA from Streptomyces lividans

Seq: 160 a.a.

Struc: 98 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1038/35079500

Nature 411:657-661 (2001)

PubMed id: 11395760

Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors.

M.Zhou, J.H.Morais-Cabral, S.Mann, R.MacKinnon.

ABSTRACT

Many voltage-dependent K+ channels open when the membrane is depolarized and then rapidly close by a process called inactivation. Neurons use inactivating K+ channels to modulate their firing frequency. In Shaker-type K+ channels, the inactivation gate, which is responsible for the closing of the channel, is formed by the channel's cytoplasmic amino terminus. Here we show that the central cavity and inner pore of the K+ channel form the receptor site for both the inactivation gate and small-molecule inhibitors. We propose that inactivation occurs by a sequential reaction in which the gate binds initially to the cytoplasmic channel surface and then enters the pore as an extended peptide. This mechanism accounts for the functional properties of K+ channel inactivation and indicates that the cavity may be the site of action for certain drugs that alter cation channel function.

Selected figure(s)

Figure 1.
Figure 1: Biophysical features of K+ channel inactivation. a, K+ currents recorded from Xenopus laevis oocytes under two-electrode voltage clamp expressing channels without an inactivation gate (1.4-IR) or with inactivation gates provided by -subunits (1.4-IR + 12). The maximum current value is 1.4 A and 2 A for noninactivating and inactivating currents, respectively. Time scale is given in b. b, K+ currents from 1.4-IR channels recorded from an excised, inside-out patch under voltage clamp in the absence (1.4-IR) or presence of 10 M TBA (+ TBA). c, Composite model of a voltage-dependent K+ channel16. The -subunit is shown in blue and the -subunit in red. The pore is represented by the KcsA K+ channel20 and the T1- complex is from ref. 16. The structures of the voltage sensor (S1-S4) and linker (T1-S1) connecting the voltage sensors to the T1 domain are unknown. An N-terminal inactivation gate is shown entering a lateral opening to gain access to the pore. The image was prepared by Molscript30 and raster-3D^31. d, Sequence alignment shows inactivation gates from K[v] 1.1 (accession number CAA 50000), Shaker B (accession number CAA 29917) and K[v]3.4 (accession number XP_002146).

Figure 2.
Figure 2: Mutational analysis of the inactivation gate-receptor interaction. a, Top, inactivation rates in K[v]1.4-IR + 12 channels determined by analysis of currents during a depolarizing pulse from -80 mV to +60 mV and recovery of current during a paired-pulse protocol7. [on] (5.0 0.3 ms) is the short time constant of a double exponential fit to current inactivation (red line) and [off] (11 0.7 s) is the time constant describing recovery in paired pulses (black line). Bottom, alanine-scanning mutagenesis of the inactivation gate. K[d], defined as [on]/ [off], was determined for K[v]1.4-IR + 12 channels with mutations to alanine or valine (position 6) at positions 2-21 in the 12 inactivation gate. The K[d] values, normalized by that for wild type, are shown. Error bars represent s.e.m. from 5 oocytes. b, Top, current recorded from an excised, inside-out patch containing 1.4-IR channels without (1.4-IR) and with (+ 4mer) a peptide corresponding to the first four amino acids of the 12 inactivation gate. Bottom, dose-response curve showing current inhibition by the 4mer peptide as a function of concentration in units of optical density volume. Data were collected from 12 patches. c, Alanine-scanning mutagenesis of pore-lining residues. The K[d] for six pore-lining mutations to alanine, normalized by the wild-type K[d], is shown. Error bars represent s.e.m. from 3-7 oocytes. d, Double-mutant cycle analysis between pore-lining residues and residues on the inactivation gate. calculated for six residues on the inactivation gate and four residues on the pore-lining helix is shown. Inactivation did not occur when Y569A on K[v]1.4-IR was paired with Q2A on 12. The approximate K[d] determination for V551A and I554A mutations did not allow determination of . Error bars show the s.e.m. measured in 5 oocytes. e, Summary of mutational analysis. Left, two diagonally positioned KcsA K+ channel subunits are shown in C[ ]trace, with pore-lining residues of the KcsA K+ channel shown as sticks but labelled according to K[v]1.4 residue numbering. Right, an extended strand model for the first six residues of the inactivation gate with side chains shown as sticks. Green and purple connecting lines identify coupled residues in the mutant cycle analysis.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2001, 411, 657-661) copyright 2001.

Figures were selected by an automated process.