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      The Desensitization Gating of the MthK K + Channel Is Governed by Its Cytoplasmic Amino Terminus

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      PLoS Biology

      Public Library of Science

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          Abstract

          The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance ( 1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

          Author Summary

          Nerve cells use ion channels, pores in the cell membrane, to send messages in the form of electrical signals between cells. Most ion channels have evolved several elaborate mechanisms that allow the channels to close quickly after opening to prevent wasteful leakage of the electrochemical potential—the currency of neuron communication—across the cell membrane. The process is known as inactivation or desensitization. Previous study on the model RCK-containing MthK K + channel in the enlarged Escherichia coli membrane has shown that this archaeon channel also undergoes desensitization. Using the same method, we demonstrate that the desensitization is indeed an intrinsic molecular property of the MthK protein. We show that a specific region of MthK, the short N terminus of the protein, functions as a structurally independent domain and is entirely responsible for the desensitization gating process. Moreover, we show that this N-terminal domain interacts with the C-terminal RCK domain as part of the desensitization mechanism. This unique desensitization mechanism, by interaction between the two cytoplasmic termini, is distinct from those traditional mechanisms known as N- and C-type inactivation found in many voltage-gated Na + and K + channels or as the desensitization observed in the glutamate receptors. Since the KTN/RCK domain is found in a large number of prokaryotic K + channels and transporters, this unique mechanism may be common to these transport systems for regulating the K + flux through the cell membrane.

          Abstract

          The N terminus of the ion channel MthK functions as a structurally independent domain and is entirely responsible for the desensitization gating process required for neuron-to-neuron communication.

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          Most cited references 38

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          Crystal structure of a mammalian voltage-dependent Shaker family K+ channel.

          Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.
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            Biophysical and molecular mechanisms of Shaker potassium channel inactivation.

            The potassium channels encoded by the Drosophila Shaker gene activate and inactivate rapidly when the membrane potential becomes more positive. Site-directed mutagenesis and single-channel patch-clamp recording were used to explore the molecular transitions that underlie inactivation in Shaker potassium channels expressed in Xenopus oocytes. A region near the amino terminus with an important role in inactivation has now been identified. The results suggest a model where this region forms a cytoplasmic domain that interacts with the open channel to cause inactivation.
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              Crystal structure and mechanism of a calcium-gated potassium channel.

              Ion channels exhibit two essential biophysical properties; that is, selective ion conduction, and the ability to gate-open in response to an appropriate stimulus. Two general categories of ion channel gating are defined by the initiating stimulus: ligand binding (neurotransmitter- or second-messenger-gated channels) or membrane voltage (voltage-gated channels). Here we present the structural basis of ligand gating in a K(+) channel that opens in response to intracellular Ca(2+). We have cloned, expressed, analysed electrical properties, and determined the crystal structure of a K(+) channel (MthK) from Methanobacterium thermoautotrophicum in the Ca(2+)-bound, opened state. Eight RCK domains (regulators of K(+) conductance) form a gating ring at the intracellular membrane surface. The gating ring uses the free energy of Ca(2+) binding in a simple manner to perform mechanical work to open the pore.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                pbio
                plbi
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                October 2008
                28 October 2008
                : 6
                : 10
                Affiliations
                Structural Biology Laboratory, The Salk Institute, La Jolla, California, United States of America
                University of Texas, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: choe@ 123456salk.edu
                Article
                08-PLBI-RA-0543R3 plbi-06-09-05
                10.1371/journal.pbio.0060223
                2573919
                18959476
                Copyright: © 2008 Kuo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 11
                Categories
                Research Article
                Biochemistry
                Biophysics
                Microbiology
                Neuroscience
                Custom metadata
                Kuo MM-C, Maslennikov I, Molden B, Choe S (2008) The desensitization gating of the MthK K + channel is governed by its cytoplasmic amino terminus. PLoS Biol 6(10): e223. doi: 10.1371/journal.pbio.0060223

                Life sciences

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