scholarly journals Voltage Sensing in Bacterial Protein Translocation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 78 ◽  
Author(s):  
Denis G. Knyazev ◽  
Roland Kuttner ◽  
Ana-Nicoleta Bondar ◽  
Mirjam Zimmerman ◽  
Christine Siligan ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Bacterial Protein ◽  
Voltage Sensing ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Dipolar Orientation ◽  
Polypeptide Chains ◽  
Gating Charges

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

scholarly journals The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation

2021 ◽  
Vol 153 (11) ◽  
Author(s):  
Nicoletta Savalli ◽  
Marina Angelini ◽  
Federica Steccanella ◽  
Julian Wier ◽  
Fenfen Wu ◽  
...  
Keyword(s):  
Critical Role ◽  
Ionic Current ◽  
Voltage Sensor ◽  
The Other ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Voltage Sensors ◽  
Voltage Dependent ◽  
Rapid Activation

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of CaV1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both CaV1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human CaV1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in CaV1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca2+ release. The prominent role of VSD-I in governing CaV1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished CaV1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of CaV activation, which accounted for both time- and voltage-dependent properties of CaV1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how CaV1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human CaV1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.

scholarly journals The BK channel gating ring is strongly coupled to the voltage sensor

2018 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Divalent Ions ◽  
Gating Currents ◽  
Open Probability ◽  
Voltage Dependent ◽  
Tightly Coupled

ABSTRACTThe open probability of large conductance voltage- and calcium-dependent potassium (BK) channels is regulated allosterically by changes in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). The divalent cation sensors reside within the gating ring formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each of the four channel subunits. Overall, the gating ring contains 12 sites that can bind Ca2+ with different affinities. Using patch-clamp fluorometry, we have shown robust changes in FRET signals within the gating ring in response to divalent ions and voltage, which do not directly track open probability. Only the conformational changes triggered through the RCK1 binding site are voltage-dependent in presence of Ca2+. Because the gating ring is outside the electric field, it must gain voltage sensitivity from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of voltage sensor dynamics known to shift gating currents produce a cognate shift in the gating ring voltage dependence, whereas changing BK channels’ relative probability of opening had little effect. These results strongly suggest that the conformational changes of the RCK1 domain of the gating ring are tightly coupled to the voltage sensor function, and this interaction is central to the allosteric modulation of BK channels.

scholarly journals Nonsensing residues in S3–S4 linker’s C terminus affect the voltage sensor set point in K+ channels

2018 ◽  
Vol 150 (2) ◽  
pp. 307-321 ◽  
Author(s):  
Joao L. Carvalho-de-Souza ◽  
Francisco Bezanilla
Keyword(s):  
Intermediate State ◽  
Voltage Dependence ◽  
State Model ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
K Channels ◽  
C Terminus ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Three State Model

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non–voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence–related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman’s coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non–voltage-sensing residues on VSD dynamics.

scholarly journals Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

2016 ◽  
Vol 113 (27) ◽  
pp. 7521-7526 ◽  
Author(s):  
Souhei Sakata ◽  
Yuka Jinno ◽  
Akira Kawanabe ◽  
Yasushi Okamura
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Conformational Changes ◽  
Unnatural Amino Acid ◽  
Voltage Sensor ◽  
Voltage Sensing ◽  
Cytoplasmic Region ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

scholarly journals Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Pablo Miranda ◽  
Miguel Holmgren ◽  
Teresa Giraldez
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Cation Binding ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Calcium Dependent ◽  
Transmembrane Voltage ◽  
Voltage Dependent

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

scholarly journals The sodium leak channel complex is modulated by voltage and extracellular calcium

2019 ◽  
Author(s):  
Han Chow Chua ◽  
Matthias Wulf ◽  
Claudia Weidling ◽  
Lise Pilgaard Rasmussen ◽  
Stephan Alexander Pless
Keyword(s):  
Neuronal Excitability ◽  
Divalent Cations ◽  
Voltage Sensitivity ◽  
List Type ◽  
Monovalent Cations ◽  
Voltage Sensing ◽  
Leak Channel ◽  
Voltage Dependent ◽  
Life Threatening ◽  
Constitutively Active

SummaryThe sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that the robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80 and FAM155A. The resulting NALCN channel complex is constitutively active, conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca2+. Despite the smaller number of voltage-sensing residues in the putative voltage sensors of NALCN, the channel complex shows voltage-dependent modulation of the constitutive current, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points towards formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting.HighlightsFunction of NALCN requires UNC79, UNC80 and FAM155AThe complex is permeable to monovalent cations, but is blocked by divalent cationsThe complex displays a constitutively active, voltage-modulated current phenotypePositively charged side chains in S4 of NALCN VSD I and II confer voltage sensitivity

scholarly journals Voltage Sensitivity of the Bacterial Protein Translocation Channel

2016 ◽  
Vol 110 (3) ◽  
pp. 354a
Author(s):  
Denis G. Knyazev ◽  
Roland Kuttner ◽  
Christine Siligan ◽  
Lukas Winter ◽  
Peter Pohl
Keyword(s):  
Protein Translocation ◽  
Voltage Sensitivity ◽  
Bacterial Protein

scholarly journals Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape

2018 ◽  
Vol 150 (7) ◽  
pp. 1017-1024 ◽  
Author(s):  
Daniel T. Infield ◽  
Elizabeth E.L. Lee ◽  
Jason D. Galpin ◽  
Grace D. Galles ◽  
Francisco Bezanilla ◽  
...  
Keyword(s):  
Positive Charge ◽  
Voltage Sensor ◽  
Nonsense Suppression ◽  
Side Chain ◽  
Voltage Sensitivity ◽  
Shaker Potassium Channel ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Kinetics Of

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance–voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

scholarly journals Engineering an enhanced voltage-sensing phosphatase

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Akira Kawanabe ◽  
Natsuki Mizutani ◽  
Onur K. Polat ◽  
Tomoko Yonezawa ◽  
Takafumi Kawai ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Danio Rerio ◽  
Mammalian Cells ◽  
Living Cells ◽  
Voltage Sensing ◽  
Phosphatase Domain ◽  
Molecular Tool ◽  
Voltage Dependent ◽  
Membrane Spanning

Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.

scholarly journals Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Cav2.3 calcium channels

2012 ◽  
Vol 139 (3) ◽  
pp. 219-234 ◽  
Author(s):  
Aleksandr Shcheglovitov ◽  
Iuliia Vitko ◽  
Roman M. Lazarenko ◽  
Peihan Orestes ◽  
Slobodan M. Todorovic ◽  
...  
Keyword(s):  
Trace Metals ◽  
Voltage Dependence ◽  
Voltage Sensitivity ◽  
Amino Acid Residues ◽  
Voltage Range ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Charge Movements ◽  
The Brain ◽  
Gating Charges

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Cav2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1–IS2 (H111) and IS2–IS3 (H179 and H183) loops potentiates Cav2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Cav2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the “ON” position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Cav2.3, potentially affecting synaptic transmission and plasticity in the brain.

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