Heme impairs the ball-and-chain inactivation of potassium channels (original) (raw)
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Impact of intracellular hemin on N-type inactivation of voltage-gated K+ channels
Pflügers Archiv - European Journal of Physiology
N-type inactivation of voltage-gated K + channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary β subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K + channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvβ1.1, Kvβ1.2, and Kvβ3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvβ1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC 50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary β subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation. Keywords K + channel inactivation. A-type channel. β subunit. Hemin. Heme. Patch clamp
A heme-binding domain controls regulation of ATP-dependent potassium channels
Proceedings of the National Academy of Sciences of the United States of America, 2016
Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATPchannels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.
Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel
Nature Structural & Molecular Biology, 2017
C-type inactivation underlies important roles played by voltage-gated K + (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here, we report that in voltage-gated Shaker K + channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K + site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K + site, which is formed by eight carbonyl oxygen atoms, appears to be slightly too small to readily accommodate a K + ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark characteristic of C-type inactivation. Kv channels underlie the repolarization phase of the action potential in excitable cells including nerve and heart cells. The channel activation gate is controlled by membrane voltage such that it opens upon membrane depolarization and closes on hyperpolarization 1, 2. However, even when depolarization is maintained and the activation gate remains open, most Kv channels still enter a nonconducting state, a process called inactivation. Two mechanistically distant types of inactivation are commonly recognized 3-5 : N-type inactivation, which results from occlusion of the channel's ion pore by the Nterminus of either the channel protein itself or its (auxiliary) β subunit 4-6 , and C-type inactivation, whose mechanistic interpretation presently remains controversial. C-type inactivation enables Kv channels to perform important tasks such as shaping cardiac action potentials to allow sufficient Ca 2+ influx to trigger effective myocyte contraction and
The Journal of General Physiology, 1999
Kv4 channels represent the main class of brain A-type K+channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996.J. Neurophysiol.75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997.Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993.J. Gen. Physiol.101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994.Biophys. J.67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region ...
N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide
Journal of Biological Chemistry, 2008
The effects of the inactivating peptide from the eukaryotic Shaker B K ؉ channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr 8 , which exhibit the strongest STD effects; the C4H in the imidazole ring of His 16 ; the methyl protons of Val 4 , Leu 7 , and Leu 10 and the side chain amine protons of one, if not both, the Lys 18 and Lys 19 residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val 4 and Leu 10 , whereas His 16 seems similarly affected as before. Conversely, STD effects on Tyr 8 are strongly diminished, and those on Lys 18 and/or Lys 19 are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of 13 C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a -hairpin structure when bound to the KcsA channel. Indeed, docking such a -hairpin structure into an open pore model for K ؉ channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.
Conformational Changes in a Mammalian Voltage-Dependent Potassium Channel Inactivation Peptide
Biochemistry, 1998
Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (K V ) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic K V 3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no R-helix or -sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial -sheet structure. Oxidation of the K V 3.4 ball peptide facilitates formation of a disulfide bond between Cys 6 and Cys 24 and adoption of a partial -sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt -sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the K V 3.4 ball peptide structure predicts a -hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant K V 3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt -structure whatever the environment, and regardless of oxidation state. The results suggest that the K V 3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the K V 3.4 ball domain in vivo.
Molecular and functional properties of two-pore-domain potassium channels
American Journal of Physiology-Renal Physiology, 2000
The two-pore-domain K+ channels, or K2P channels, constitute a novel class of K+channel subunits. They have four transmembrane segments and are active as dimers. The tissue distribution of these channels is widespread, and they are found in both excitable and nonexcitable cells. K2P channels produce currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K+ channel blockers. These properties designate them as background K+ channels. They are expected to play a major role in setting the resting membrane potential in many cell types. Another salient feature of K2P channels is the diversity of their regulatory mechanisms. The weak inward rectifiers TWIK-1 and TWIK-2 are stimulated by activators of protein kinase C and decreased by internal acidification, the baseline TWIK-related acid-sensitive K+ (TASK)-1 and TASK-2 channels are sensitive to external pH changes in a narrow ra...
Neuron, 1994
Cyclic nucleotide-gated (CNG) channels in photoreceptors and olfactory neurons are activated by intracellular ligands (cAMP and cGMP) rather than voltage. Surprisingly, these channels share amino acid sequence homology with voltage-gated channels. Here we show that the distinct gating mechanisms exhibited by CNG and voltage-gated channels share features that reflect this structural homology. Thus, a 20 amino acid peptide ("ball peptide") derived from the Shaker-type K + channel and responsible for its rapid inactivation also blocks CNG channels. Moreover, the peptide selectively blocks open CNG channels and prevents channel closure, showing that CNG channel activation, like activation of voltagedependent K + channels, involves the opening of a gate located on the intracellular side of the peptide-binding site. Amino acid substitutions in the peptide cause similar changes in blocking affinity of CNG and K + channels, suggesting a conserved binding site. Using a chimeric retinal/olfactory channel, we show that the difference in the peptide affinity of the two CNG channels is due to a difference in the amino acid sequence of the conserved pore-forming region, demonstrating that this domain forms part of the peptide receptor.
Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain
The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 ␣-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1-3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a͞KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific ''ball'' domain of the Kv1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.