Conformational Changes in a Mammalian Voltage-Dependent Potassium Channel Inactivation Peptide (original) (raw)
Related papers
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.
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.
State-dependent Block of BK Channels by Synthesized Shaker Ball Peptides
The Journal of General Physiology, 2006
Crystal structures of potassium channels have strongly corroborated an earlier hypothetical picture based on functional studies, in which the channel gate was located on the cytoplasmic side of the pore. However, accessibility studies on several types of ligand-sensitive K+channels have suggested that their activation gates may be located near or within the selectivity filter instead. It remains to be determined to what extent the physical location of the gate is conserved across the large K+channel family. Direct evidence about the location of the gate in large conductance calcium-activated K+(BK) channels, which are gated by both voltage and ligand (calcium), has been scarce. Our earlier kinetic measurements of the block of BK channels by internal quaternary ammonium ions have raised the possibility that they may lack a cytoplasmic gate. We show in this study that a synthesized Shaker ball peptide (ShBP) homologue acts as a state-dependent blocker for BK channels when applied inte...
Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel
Biophysical Journal, 1995
The conformation of the inactivating peptide of the ShakerB K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt structure and the observation that trypsin hydrolysis of the ShB peptide prevents both structure formation and channel inactivation lend further support to the hypothesis that adoption of 1B structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.
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.
The Journal of General Physiology, 1999
The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K d, 16.5 nM) and BPTI (K d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.
Journal of Biological Chemistry, 1998
Certain -subunits exert profound effects on the kinetics of voltage-gated (K v) potassium channel inactivation through an interaction between the amino-terminal "inactivation domain" of the -subunit and a "receptor" located at or near the cytoplasmic mouth of the channel pore. Here we used a bacterial random peptide library to examine the structural requirements for this interaction. To identify peptides that bind K v 1.1 we screened the library against a synthetic peptide corresponding to the predicted S4-S5 cytoplasmic loop of the K v 1.1 ␣-subunit (residues 313-328). Among the highest affinity interactors were peptides with significant homology to the amino terminus of K v 1. We performed a second screen using a peptide from the amino terminus of K v 1 (residues 2-31) as "bait" and identified peptide sequences with significant homology to the S4-S5 loop of K v 1.1. A series of synthetic peptides containing mutations of the wild-type K v 1 and K v 1.1 sequences were examined for their ability to inhibit K v 1/K v 1.1 binding. Amino acids Arg 20 and Leu 21 in K v 1 and residues Arg 324 and Leu 328 in K v 1.1 were found to be important for the interaction. Taken together, these data provide support for the contention that the S4-S5 loop of the K v 1.1 ␣ subunit is the likely acceptor for the K v 1 inactivation domain and provide information about residues that may underlie the protein-protein interactions responsible for -subunit mediated K v channel inactivation. Voltage-gated (K v) 1 K ϩ channels are heteromeric protein complexes composed of four integral membrane ion-conducting ␣-subunits and four cytoplasmic -subunits. These channels are critical for action potential conduction and neurotransmitter release and are fundamental to the control of neuronal excitability (1-5). The diversity of function of K ϩ channels is reflected in the heterogeneity of channels that have been found in the nervous system, both in terms of electrophysiological properties and in the multitude of genes that encode them (6-11, 32). Electrophysiological studies have broadly classified K v channels into two types: delayed-rectifier channels, which are characterized by their slow inactivation, and A-type K v channels, which inactivate rapidly. Although the majority of K v channel ␣-subunit cDNAs give rise to delayed-rectifier-type currents when expressed in heterologous cells, it has been
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
Fast Inactivation of Voltage-Gated K+ Channels: From Cartoon to Structure
Physiology
Fast inactivation of voltage-gated potassium (Kv) channels is the best understood gating transition in ion channels and is brought about by an NH2-terminal domain (ball domain) of the channel’s α-subunit, which physically blocks the open pore. Recent analysis by nuclear magnetic resonance spectroscopy showed that ball domains from various Kv channels exhibit well-defined but distinct structures in aqueous solution.