Structural dynamics determine voltage and pH gating in human voltage-gated proton channel

AbstractVoltage-gated ion channels are key players of electrical signaling in cells. As a unique subfamily, voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. They are gated by both voltage and transmembrane proton gradient (i.e ΔpH), serving as acid extruders in most cells. Amongst their many functions, Hv channels are known to regulate the intracellular pH of human spermatozoa and compensate for the charge and pH imbalances caused by NADPH oxidases in phagocytes. Like the canonical voltage sensors, the Hv channel is a bundle of 4 helices (named S1 through S4), with the S4 segment carrying 3 positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels generally agree on an outwards movement of the S4 segment upon activating voltage, but the real time conformational transitions are still unattainable. With purified human voltage-gated proton (hHv1) channel reconstituted in liposomes, we have examined its conformational dynamics at different voltage and pHs using the single molecule fluorescence resonance energy transfer (smFRET). Here we provided the first glimpse of real time conformational trajectories of the hHv1 voltage sensor and showed that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results suggested the biological gating is determined by the conformational distributions of the hHv1 voltage sensor, rather than the conformational transitions between the presumptive ‘resting’ and ‘activated’ conformations. We further identified H140 as the key residue sensing extracellular pH and showed that both the intracellular and extracellular pH sensors act on the voltage sensing S4 segment to enrich the resting conformations. Taken together, we proposed a model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework to understand the voltage sensing and gating in other voltage-gated ion channels.

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Immobilizing the Moving Parts of Voltage-Gated Ion Channels

The Journal of General Physiology ◽

10.1085/jgp.116.3.461 ◽

2000 ◽

Vol 116 (3) ◽

pp. 461-476 ◽

Cited By ~ 97

Author(s):

Richard Horn ◽

Shinghua Ding ◽

Hermann J. Gruber

Keyword(s):

Ion Channels ◽

Potassium Channels ◽

Sodium Channel ◽

Ultraviolet Irradiation ◽

Voltage Sensors ◽

Voltage Gated ◽

Voltage Gated Ion Channels ◽

S4 Segment ◽

Moving Parts ◽

Gated Ion Channels

Voltage-gated ion channels have at least two classes of moving parts, voltage sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To explore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzophenone-4-carboxamidocysteine methanethiosulfonate, that can be tethered to cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium channel. The benzophenone-labeled inactivation gate of the sodium channel can be trapped selectively either in an open or closed state by ultraviolet irradiation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As predicted by the tetrameric stoichiometry of these potassium channels, ultraviolet irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the movements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depolarization moves the benzophenone adduct out of a restricted environment. Immobilization of the S4 segment of the second domain of sodium channels prevents channels from opening. By contrast, photocross-linking the S4 segment of the fourth domain of the sodium channel has effects on both activation and inactivation. Our results indicate that specific voltage sensors of the sodium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4, is at least a two-step process, each step coupled to a different gate.

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Real-Time Kinetic Modeling of Voltage-Gated Ion Channels Using Dynamic Clamp

Biophysical Journal ◽

10.1529/biophysj.107.118190 ◽

2008 ◽

Vol 95 (1) ◽

pp. 66-87 ◽

Cited By ~ 55

Author(s):

Lorin S. Milescu ◽

Tadashi Yamanishi ◽

Krzysztof Ptak ◽

Murtaza Z. Mogri ◽

Jeffrey C. Smith

Keyword(s):

Ion Channels ◽

Real Time ◽

Kinetic Modeling ◽

Dynamic Clamp ◽

Voltage Gated ◽

Voltage Gated Ion Channels ◽

Gated Ion Channels

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Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology

eLife ◽

10.7554/elife.52164 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Xiao Tao ◽

Roderick MacKinnon

Keyword(s):

Ion Channels ◽

Conformational Changes ◽

Voltage Sensor ◽

Structural Domains ◽

Structural Class ◽

Voltage Sensors ◽

Voltage Gated ◽

Voltage Dependent ◽

Voltage Gated Ion Channels ◽

Gated Ion Channels

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

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