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RNA DNA Protein Alternative splicing RNA editing Allosteric modulators, e.g., Ca 2+ /calmodulin, G-proteins, nucleotides Plasma membrane NUCLEUS Associated subunits, e.g., Kir6 + SUR, KCNQ + KCNE, AMPAR + stargazin Type III Mechanisms of Gating Type I Type II Type III K v 1.2 TRPV1 Kir2.2 KcsA TRAAK ASIC1 Piezo1 MscL ClC-K AMPAR nAChR GABA A R NMDAR CNG TAX-4 GLIC TMEM16 Na v Pas Ca v 1.1 HCN1 Co- and post-translational modification, e.g., phosphorylation, glycosylation, acetylation, methylation, palmitoylation, ubiquitylation, SUMOylation, proteolytic cleavage Epigenetics Adaptor and cell signaling proteins, e.g., PSD-95, AKAPs, G-proteins Other modulatory compounds, e.g., ions, ligands, toxins, drugs RNA binding proteins, e.g., FMRP Mechanisms of Regulation H v 1 P2X3 p H M e c h a n o V o l t a g e L i g a n d T e m p e r a t u r e KcsA ASIC ENaC/Deg MscS/L Piezo VRAC CaCC CLC K 2P TRP TPC H v K v 1-9 Na v Ca v K v 10-12 HCN CNG K Ca Kir P2X CFTR RyR IP 3 R CRAC NMDAR kainate AMPAR nAChR 5-HT 3 R GABA A R GlyR GLIC ZAC Includes voltage-, mechanical,- and temperature- gated channels involved in recognition of physical stimuli such as membrane voltage, tension/curvature, fluid flow, and temperature Discrete or delocalized sensors are not constrained by stereochemistry and therefore may not require conserved structural domains Convergent evolution of function pH-gated channels involved in recognition of cellular acidity Discrete or delocalized sensors may or may not be conserved Many functional groups are pH titratable, including protein backbones Primarily ligand-gated channels involved in recognition of chemical stimuli such as small molecule ligands/proteins Sensors are conserved and discrete structural modules Derived from common ancestral proteins (amenable to phylogenetic analysis) Transcription Translation Trafficking Subunit/Complex assembly Lipids, e.g., cholesterol, sphingomyelin, PIP 2 Co-translational assembly Type II Type I See online version for legends and references 594 Cell 170, July 27, 2017 © 2017 Published by Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2017.07.019 SnapShot: Channel Gating Mechanisms Marcel P. Goldschen-Ohm 1 and Baron Chanda 1,2 1 Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53706, USA 2 Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA

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Page 1: Chanda Lab – Structure and Dynamics of Channels ...bclab.neuro.wisc.edu/wp-content/uploads/All Publications...Ion channel families are broadly classified into three types according

RNADNA Protein

Alternativesplicing

RNAediting

Allosteric modulators,

e.g., Ca2+/calmodulin,

G-proteins, nucleotides Plasma membraneN U C L E U S

Associated subunits,

e.g., Kir6 + SUR,

KCNQ + KCNE,

AMPAR + stargazin

Type III

Mechanisms of Gating

Type I Type II Type III

Kv1.2

TRPV1

Kir2.2

KcsA

TRAAK

ASIC1

Piezo1

MscL

ClC-K

AMPAR

nAChR

GABAAR

NMDAR

CNG TAX-4

GLIC

TMEM16

NavPasCav1.1

HCN1

Co- and post-translational modi�cation,

e.g., phosphorylation, glycosylation, acetylation,

methylation, palmitoylation, ubiquitylation,

SUMOylation, proteolytic cleavage

Epigenetics

Adaptor and cell

signaling proteins,

e.g., PSD-95, AKAPs,

G-proteins

Other modulatory

compounds,

e.g., ions, ligands,

toxins, drugs

RNA binding

proteins,

e.g., FMRP

Mechanisms of Regulation

Hv1

P2X3

pHMechano

Voltage

Liga

nd

Temperat

ure

Kc

sA

AS

ICE

NaC

/Deg

Msc

S/L

Piezo

VRAC

CaCC

CLC

K2P

TRP

TPC

Hv

Kv 1

-9

Na

v

Ca v

K v10

-12

HCN

CNG

KC

a

Kir

P2X

CFTR

RyR

IP3R

CRAC

NMDAR

kainate

AMPAR

nAChR

5-HT

3 RG

AB

AA R

GlyR

GL

IC

ZA

C

Includes voltage-, mechanical,- and temperature-gated channels involved in recognition of physical stimuli such as membrane voltage, tension/curvature, �uid �ow, and temperature

Discrete or delocalized sensors are not constrained by stereochemistry and therefore may not require conserved structural domains

Convergent evolution of function

pH-gated channels involved in recognition of cellular acidity

Discrete or delocalized sensors may or may not be conserved

Many functional groups are pH titratable, including protein backbones

Primarily ligand-gated channels involvedin recognition of chemical stimuli such as small molecule ligands/proteins

Sensors are conserved and discrete structural modules

Derived from common ancestral proteins (amenable to phylogenetic analysis)

Transcription Translation Traf�cking

Subunit/Complex assembly

Lipids,

e.g., cholesterol,

sphingomyelin, PIP2

Co-translational

assembly

Type II

Type I

See online version for legends and references594 Cell 170, July 27, 2017 © 2017 Published by Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2017.07.019

SnapShot: Channel Gating MechanismsMarcel P. Goldschen-Ohm1 and Baron Chanda1,2

1Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53706, USA2Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA

Page 2: Chanda Lab – Structure and Dynamics of Channels ...bclab.neuro.wisc.edu/wp-content/uploads/All Publications...Ion channel families are broadly classified into three types according

SnapShot: Channel Gating MechanismsMarcel P. Goldschen-Ohm1 and Baron Chanda1,2

1Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53706, USA2Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA

Mechanisms of GatingIon channel families are broadly classified into three types according to their major mechanisms of activation. Type I comprises primarily ligand-gated channels. These

channels are distinguished by the requirement for conserved structural domains that act as recognition sites for specific ligand molecules and whose binding energy is used to gate the channel pore. Exemplar members include pentameric (cys loop) and ionotropic glutamate neurotransmitter receptors (Plested, 2016); nucleotide-gated channels such as Kir, including KATP and GIRK family members P2X and CNG (Craven and Zagotta, 2006); and protein-gated channels such as ORAI+STIM. Type II includes proton-gated channels whose activity responds to changes in the protonation state of titratable groups either localized in discrete functional domains or distributed throughout the protein. Unlike channels belonging to type I, the residues comprising these sites do not require a specific stereochemistry to match a particular ligand. Examples include ASICs (Bos-cardin et al., 2016) and K2P family members such as TRASK and TALK, as well as prokaryotic channels GLIC and KcsA (Cuello et al., 2010). Type III includes channels gated by physical stimuli such as voltage, temperature, or mechanical stress. Sensors of physical stimuli are not constrained by stereochemistry and may or may not consist of conserved structural domains. This lack of conservation may reflect, at a fundamental level, the diversity of molecular mechanisms in sensing physical stimulus that may have evolved independently in different branches of the phylogenetic tree. Voltage-gated channels include those that have a canonical voltage sensing domain (VSD) such as Kv, Nav, Cav, HCN, BK, TRP, and Hv (Palovcak et al., 2014), as well as channels that lack a VSD such as CLC and K2P (e.g., TREK and TRAAK; Schewe et al., 2016), suggesting that voltage sensitivity can be conferred by multiple mechanisms. Both temperature- and mechano-gated channels are structurally diverse, including members from TRP subfami-lies TRPV, TRPM, TRPC, and TRPA; K2P family members TREK and TRAAK; Hv; and the calcium-activated chloride channels (CaCCs) such as Anoctamin (TMEM16) (Palkar et al., 2015). Moreover, mechano-sensitive channels that respond to membrane tension/curvature, osmotic shock, or shear stress from fluid flow exist such as Piezo (Coste et al., 2010), MscS, and MscL, volume-regulated anion channels (VRAC), and members of the ENaC/Degenerin family of epithelial sodium channels.

Activating stimuli for channel families are indicated in a wheel diagram (center) surrounded by example structures of channels from each type (transmembrane domains are colored cyan, and extracellular and intracellular domains are colored magenta, with intracellular domains oriented toward the center). Low-resolution flexible regions are omit-ted from the structures. For Hv1, a single subunit constituting the minimal functional unit of a dimeric channel is shown. Note that, in some cases, a subset of channels within a family may not respond to a particular stimulus. The list of channels and exemplar structures is not comprehensive but includes key examples of eukaryotic as well as some widely studied prokaryotic channels that span the three types of gating mechanisms. It is important to note that all channels respond to physical stimuli such as tempera-ture, mechanical stress, or voltage to some degree and that the gating behavior of many channels is modulated by pH or binding of small molecules. Here, we sought to limit our classification to those central stimuli that mediate channel activation rather than modulation. Nonetheless, we acknowledge that this distinction is not always clear, and whether a stimulus is modulatory or activating may depend on its actions within a physiological context. Finally, we expect that this classification will evolve as we continue to explore the physical mechanisms that govern ion channel activity. PDB codes for the displayed structures are 3KG2 (AMPAR), 2QTS (ASIC1), 5GJV (Cav1.1), 5TQQ (CLC-K), 5H3O (CNG TAX-4), 4COF (GABAAR), 4HFI (GLIC), 5U6P (HCN1), 3WKV (Hv1), 3PJS (KcsA), 3SPI (Kir2.2), 2A79 (Kv1.2), 3HZQ (MscL), 5KXI (nAChR), 5X0M (NavPas), 4TLL (NMDAR), 3JAC (Piezo1), 5SVK (P2X3), 4WIS (TMEM16), 3UM7 (TRAAK), and 3J5P (TRPV1).

Mechanisms of RegulationRegulation of channel function and expression can occur at various steps from transcription and translation of the genetic code to trafficking and modulation of channel

complexes in the plasma membrane. For example, regulated mRNA splicing can result in alternative variants whose RNA messages may code for channels with differing func-tions (Meredith, 2015). Editing of RNA prior to being translated can similarly result in recoding of proteins with distinct functions. Following translation from mRNA to protein, channel subunits fold and co-assemble to form channel complexes that are trafficked to the plasma membrane (Deutsch, 2003). During these processes, channel proteins may undergo a variety of co- and post-translational modifications that can affect channel function. Some prevalent modifications include phosphorylation, glycosylation, ubiq-uitinylation, acetylation, and methylation of individual residues. For example, reversible phosphorylation of specific side chains by protein kinase and phosphatase enzymes is an important regulatory mechanism for many ion channels. Furthermore, scaffolding proteins (e.g., PSD-95 and AKAPs) cluster channels in the vicinity of cell signaling proteins important for channel regulation, such as protein kinases.

Many channel families contain a variety of distinct subunit subtypes, some of which can assemble in multiple heterogeneous combinations with differing functional proper-ties. Functional channels can also be part of a larger signaling complex that includes associated proteins that do not directly contribute to formation of the channel pore but instead alter the channel’s gating behavior or localization. For example, ATP-gated potassium channels consist of channel-forming Kir6 subunits and associated SUR sub-units, whereas association of KCNQ1 and KCNE1 subunits underlies an important potassium current in the heart. Furthermore, many channels also bind small molecules or proteins that act as allosteric modulators of channel gating. The calcium-binding protein calmodulin, G-proteins, and nucleotides are a few prevalent endogenous examples. Channel function can also be influenced by the lipid environment of the plasma membrane in which it resides. For example, phosphoinositides such as PIP2 are important regulators for a variety of channels (Hilgemann et al., 2001). Finally, many channels are also regulated by exogenous toxins, and drugs have made them important targets for disease therapies. The ability of channels to be regulated at multiple levels from transcription to modulation in their lipid environment provides numerous pathways for fine-tuning cellular physiology.

ACKNOWLEDGMENTS

We thank Rick Aldrich, David Clapham, Cindy Czajkowski, Katie Henzler-Wildman, Andy Meredith, Chris Miller, Eduardo Perozo, Andrew Plested, Gail Robertson, and Kenton Swartz for their helpful comments and suggestions. B.C. is funded by R01NS081293 and R01NS101723.

REFERENCES

Boscardin, E., Alijevic, O., Hummler, E., Frateschi, S., and Kellenberger, S. (2016). The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR Review 19. Br. J. Pharmacol. 173, 2671–2701.

Coste, B., Mathur, J., Schmidt, M., Earley, T.J., Ranade, S., Petrus, M.J., Dubin, A.E., and Patapoutian, A. (2010). Piezo1 and Piezo2 are essential components of distinct mechani-cally activated cation channels. Science 330, 55–60.

Craven, K.B., and Zagotta, W.N. (2006). CNG and HCN channels: two peas, one pod. Annu. Rev. Physiol. 68, 375–401.

Cuello, L.G., Cortes, D.M., Jogini, V., Sompornpisut, A., and Perozo, E. (2010). A molecular mechanism for proton-dependent gating in KcsA. FEBS Lett. 584, 1126–1132.

Deutsch, C. (2003). The birth of a channel. Neuron 40, 265–276.

Hilgemann, D.W., Feng, S., and Nasuhoglu, C. (2001). The complex and intriguing lives of PIP2 with ion channels and transporters. Sci. STKE 2001, re19.

Meredith, A.L. (2015). Alternative splicing. In Handbook of ion channels, J. Zheng and M.C. Trudeau, eds. (CRC Press), pp. 545–556.

Palkar, R., Lippoldt, E.K., and McKemy, D.D. (2015). The molecular and cellular basis of thermosensation in mammals. Curr. Opin. Neurobiol. 34, 14–19.

Palovcak, E., Delemotte, L., Klein, M.L., and Carnevale, V. (2014). Evolutionary imprint of activation: the design principles of VSDs. J. Gen. Physiol. 143, 145–156.

Plested, A.J. (2016). Structural mechanisms of activation and desensitization in neurotransmitter-gated ion channels. Nat. Struct. Mol. Biol. 23, 494–502.

Schewe, M., Nematian-Ardestani, E., Sun, H., Musinszki, M., Cordeiro, S., Bucci, G., de Groot, B.L., Tucker, S.J., Rapedius, M., and Baukrowitz, T. (2016). A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels. Cell 164, 937–949.

594.e1 Cell 170, July 27, 2017 © 2017 Published by Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2017.07.019