The Molecular Mechanism of Toxin-Induced Conformational Changes in a Potassium Channel: Relation to C-Type Inactivation

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Potassium (i.e., K + ) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K + channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K + channel inactivation and exhibits a remarkable structural plasticity that correlates to K + channel inactivation. The transmembrane helix 1 unwinds when the K + channel enters the inactivated state and rewinds during the transition to the closed state. In addition to wellcharacterized changes at the K + ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K + channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K + channel pore. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

Section: Resultsmentioning

confidence: 53%

Importance of lipid–pore loop interface for potassium channel structure and function

Cruijsen

Nand

Weingarth

et al. 2013

Self Cite

“…Indeed, strong experimental evidence points to the hydrogen bond network behind the selectivity filter as a driver of the inactivation process (12,13,15). The outer vestibule of KcsA is an important unstructured region in which toxins (37), blockers (35,38), and metal ions (16) bind and modulate the gating behavior of K + channels. Several results suggest a dynamic structural rearrangement in the outer vestibule during C-type inactivation gating in KcsA and Shaker K + channels (16,18,19).…”

Section: Discussionmentioning

confidence: 99%

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

Raghuraman

Islam

Mukherjee

et al. 2014

Significance C-type inactivation gating in K + channels plays an important role in controlling the firing patterns of excitable cells and is fundamental in determining the length and frequency of the cardiac action potential. At a molecular level, toxins, blockers, and metal ions bind to the outer vestibule and modulate the functional behavior of K + channels. Using KcsA, we show that the shuttling between the inactivated and conductive states of K + channels is accompanied by changes in local outer vestibule dynamics, in the absence of large conformational changes. The altered structural and hydration dynamics of the outer vestibule appear to be likely and important modulators of selectivity filter gating transitions in K + channels.

“…However, a Lys residue is absent from the GX n C element of the hBD-2 ␥-core motif (GTC). The current results also indicate that crotamine perturbs eukaryotic K v channels as do the charybdotoxin (23), kaliotoxin (24), and cobatoxin (25) family of scorpion toxins (26). Basic residues in such toxins form a cationic facet integrating Lys or Arg residues of their ␥-core GX n C motif (2, 3).…”

Section: Discussionmentioning

confidence: 82%

“…However, recent studies by Rizzi et al (29), using expressed Na v 1.1-1.6 ␣-subunits, did not find an interaction between crotamine and Na v channels. Further support for the interaction of defensin-like toxins with K v ion channels derives from recent structural mapping of the K v channel surface and, in some cases, toxin/channel complexes (23)(24)(25). Together, these facts support the concept that hBD-2 and crotamine may preferentially but not exclusively target respective Na v, NaK v , or other TTX-sensitive ion channelsversus K v channels-contributing to their net cytotoxic effects.…”

Section: Discussionmentioning

confidence: 85%

Selective reciprocity in antimicrobial activity versus cytotoxicity of hBD-2 and crotamine

Yount

Kupferwasser

Spisni

et al. 2009

Recent discoveries suggest cysteine-stabilized toxins and antimicrobial peptides have structure-activity parallels derived by common ancestry. Here, human antimicrobial peptide hBD-2 and rattlesnake venom-toxin crotamine were compared in phylogeny, 3D structure, target cell specificity, and mechanisms of action. Results indicate a striking degree of structural and phylogenetic congruence. Importantly, these polypeptides also exhibited functional reciprocity: (i) they exerted highly similar antimicrobial pH optima and spectra; (ii) both altered membrane potential consistent with ion channel-perturbing activities; and (iii) both peptides induced phosphatidylserine accessibility in eukaryotic cells. However, the Nav channel-inhibitor tetrodotoxin antagonized hBD-2 mechanisms, but not those of crotamine. As crotamine targets eukaryotic ion channels, computational docking was used to compare hBD-2 versus crotamine interactions with prototypic bacterial, fungal, or mammalian Kv channels. Models support direct interactions of each peptide with Kv channels. However, while crotamine localized to occlude Kv channels in eukaryotic but not prokaryotic cells, hBD-2 interacted with prokaryotic and eukaryotic Kv channels but did not occlude either. Together, these results support the hypothesis that antimicrobial and cytotoxic polypeptides have ancestral structurefunction homology, but evolved to preferentially target respective microbial versus mammalian ion channels via residue-specific interactions. These insights may accelerate development of antiinfective or therapeutic peptides that selectively target microbial or abnormal host cells.channel ͉ defensin ͉ host defense ͉ toxin