scholarly journals Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence

2010 ◽  
Vol 136 (1) ◽  
pp. 83-99 ◽  
Author(s):  
Andrew James Horne ◽  
Christian Joseph Peters ◽  
Thomas William Claydon ◽  
David Fedida
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Slow Component ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Activation Process ◽  
Dependent Manner ◽  
Gating Currents ◽  
Kv Channel ◽  
Voltage Dependent

The Kv1.2 channel, with its high resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at A291C reports two distinct rearrangements of the voltage sensor domains, and a comparative fluorescence scan of the S4 and S3–S4 linker residues in Shaker and Kv1.2 shows important differences in their emission at other homologous residues. Kv1.2 shows a rapid decrease in A291C emission with a time constant of 1.5 ± 0.1 ms at 60 mV (n = 11) that correlates with gating currents and reports on translocation of the S4 and S3–S4 linker. However, unlike any Kv channel studied to date, this fast component is dwarfed by a larger, slower quenching of TMRM emission during depolarizations between −120 and −50 mV (τ = 21.4 ± 2.1 ms at 60 mV, V1/2 of −73.9 ± 1.4 mV) that is not seen in either Shaker or Kv1.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltage-dependent manner that matches the time course of Kv1.2 ionic current deactivation. Fluorophores placed directly in S1 and S2 at I187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost when the extracellular S1–S2 linker of Kv1.2 is replaced with that of Kv1.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close.

scholarly journals Gating charge displacement in a monomeric voltage-gated proton (Hv1) channel

2018 ◽  
Vol 115 (37) ◽  
pp. 9240-9245 ◽  
Author(s):  
Emerson M. Carmona ◽  
H. Peter Larsson ◽  
Alan Neely ◽  
Osvaldo Alvarez ◽  
Ramon Latorre ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Sensor ◽  
Total Charge ◽  
Gating Currents ◽  
Voltage Gated ◽  
Central Transition ◽  
Physiological Processes ◽  
Gating Charge ◽  
Voltage Dependent

The voltage-gated proton (Hv1) channel, a voltage sensor and a conductive pore contained in one structural module, plays important roles in many physiological processes. Voltage sensor movements can be directly detected by measuring gating currents, and a detailed characterization of Hv1 charge displacements during channel activation can help to understand the function of this channel. We succeeded in detecting gating currents in the monomeric form of the Ciona-Hv1 channel. To decrease proton currents and better separate gating currents from ion currents, we used the low-conducting Hv1 mutant N264R. Isolated ON-gating currents decayed at increasing rates with increasing membrane depolarization, and the amount of gating charges displaced saturates at high voltages. These are two hallmarks of currents arising from the movement of charged elements within the boundaries of the cell membrane. The kinetic analysis of gating currents revealed a complex time course of the ON-gating current characterized by two peaks and a marked Cole–Moore effect. Both features argue that the voltage sensor undergoes several voltage-dependent conformational changes during activation. However, most of the charge is displaced in a single central transition. Upon voltage sensor activation, the charge is trapped, and only a fast component that carries a small percentage of the total charge is observed in the OFF. We hypothesize that trapping is due to the presence of the arginine side chain in position 264, which acts as a blocking ion. We conclude that the movement of the voltage sensor must proceed through at least five states to account for our experimental data satisfactorily.

scholarly journals Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

2014 ◽  
Vol 144 (5) ◽  
pp. 457-467 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Benjamin M. Haehnel ◽  
Baron Chanda
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Electromechanical Coupling ◽  
Structural Integrity ◽  
Voltage Sensor ◽  
Amino Acid Residues ◽  
Kv Channel ◽  
Electromechanical Transduction ◽  
Voltage Dependent ◽  
Graph Theoretical Approach

Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

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 The Voltage Sensor in Voltage-Dependent Ion Channels

2000 ◽  
Vol 80 (2) ◽  
pp. 555-592 ◽  
Author(s):  
Francisco Bezanilla
Keyword(s):  
Membrane Potential ◽  
Conformational Changes ◽  
Structural Information ◽  
Noise Analysis ◽  
Voltage Sensor ◽  
Fluorescence Labeling ◽  
Gating Current ◽  
Gating Currents ◽  
Gating Charge ◽  
Voltage Dependent

In voltage-dependent Na, K, or Ca channels, the probability of opening is modified by the membrane potential. This is achieved through a voltage sensor that detects the voltage and transfers its energy to the pore to control its gate. We present here the theoretical basis of the energy coupling between the electric field and the voltage, which allows the interpretation of the gating charge that moves in one channel. Movement of the gating charge constitutes the gating current. The properties are described, along with macroscopic data and gating current noise analysis, in relation to the operation of the voltage sensor and the opening of the channel. Structural details of the voltage sensor operation were resolved initially by locating the residues that make up the voltage sensor using mutagenesis experiments and determining the number of charges per channel. The changes in conformation are then analyzed based on the differential exposure of cysteine or histidine-substituted residues. Site-directed fluorescence labeling is then analyzed as another powerful indicator of conformational changes that allows time and voltage correlation of local changes seen by the fluorophores with the global change seen by the electrophysiology of gating currents and ionic currents. Finally, we describe the novel results on lanthanide-based resonance energy transfer that show small distance changes between residues in the channel molecule. All of the electrophysiological and the structural information are finally summarized in a physical model of a voltage-dependent channel in which a change in membrane potential causes rotation of the S4 segment that changes the exposure of the basic residues from an internally connected aqueous crevice at hyperpolarized potentials to an externally connected aqueous crevice at depolarized potentials.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA.

1994 ◽  
Vol 103 (5) ◽  
pp. 731-753 ◽  
Author(s):  
E A Ertel ◽  
M M Smith ◽  
M D Leibowitz ◽  
C J Cohen
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Slow Component ◽  
Ionic Current ◽  
Reversal Potential ◽  
Ca Channel ◽  
Ca Channels ◽  
Gating Currents ◽  
Voltage Range ◽  
Charge Movements

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L-type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega-Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Dendritic origin of late events in optical recordings from salamander olfactory bulb

1992 ◽  
Vol 68 (3) ◽  
pp. 786-806 ◽  
Author(s):  
A. R. Cinelli ◽  
B. M. Salzberg
Keyword(s):  
Olfactory Bulb ◽  
Time Course ◽  
Slow Component ◽  
Field Potential ◽  
Olfactory Nerve ◽  
Nerve Fibers ◽  
Dependent Manner ◽  
Gamma Aminobutyric Acid ◽  
Direct Stimulation ◽  
Compound Action

1. Optical recordings of membrane-potential changes were used to characterize the origin and properties of the electrical signals from the dendritic level in slices of the salamander olfactory bulb. 2. The optical events were correlated with field-potential waves recorded simultaneously. Both responses exhibited patterns similar to those found in other species. 3. Orthodromic stimulation evoked a compound action potential in the olfactory nerve fibers, followed by two additional principal waves (N1 and N2). These field-potential waves reflected excitatory postsynaptic potentials at the primary mitral/tufted and granule cell dendrites, respectively. 4. Extrinsic optical signals from horizontal slices stained with the pyrazo-oxonal dye RH-155 showed a characteristic sequence of depolarizing and hyperpolarizing events. All of the signals exhibited a wavelength dependence expected for this dye and were abolished in the presence of high K+ in the bath. 5. According to their time courses, depolarizing responses under normal recording conditions were divided into two components, fast and slow. Orthodromic stimuli evoked a fast presynaptic response that represents synchronous compound action potentials from olfactory nerve fibers. At subglomerular levels, additional fast responses could often be recorded at the peri/subglomerular level and in the mitral/tufted somata region. These postsynaptic responses partially coincided with the rising phase of a different depolarizing signal, a slow component characterized by its prolonged time course. 6. With orthodromic stimulation, this slow signal attained its largest amplitude in the zone between the glomeruli and the superficial part of the external plexiform layer (EPL). Antidromic stimuli evoked a signal with some similarities to the one evoked orthodromically, but originating in deeper EPL regions. 7. Slow components were characterized by their Ca dependence. Low Ca2+ medium, or calcium channel blockers, suppressed this optical component, whether evoked orthodromically, antidromically, or by direct stimulation. In addition, Ba2+ (2.5–3.6 mM) in the bath did not abolish these responses, suggesting that they do not reflect a glial depolarization in response to elevated extracellular K+ concentration ([K+]o). 8. Locally applied stimuli next to the glomerular layer elicited these signals in 5–10 microM tetrodotoxin (TTX) or in low extracellular Na+ concentration ([Na+]o) medium, but antidromic or orthodromic stimuli failed to evoke the response under these conditions. The sizes of the responses to local stimuli remained constant, but an increase in their duration was observed in either TTX or low [Na+]o. 9. gamma-Aminobutyric acid (GABA) and baclofen reduced the size of the slow components in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals R1 in the Shaker S4 occupies the gating charge transfer center in the resting state

2011 ◽  
Vol 138 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Meng-chin A. Lin ◽  
Jui-Yi Hsieh ◽  
Allan F. Mock ◽  
Diane M. Papazian
Keyword(s):  
Charge Transfer ◽  
Binding Site ◽  
Resting State ◽  
Slow Component ◽  
Maximum Amplitude ◽  
Voltage Sensor ◽  
Shaker Channels ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Arginine Residues

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1–R4) cross the transmembrane electric field. It has been proposed that R1–R4 movement is facilitated by a “gating charge transfer center” comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn2+ on channel activity. In I287H+R1H, Zn2+ generated a slow component of activation with a maximum amplitude (Aslow,max) of ∼56%, indicating that only a fraction of voltage sensors can bind Zn2+ at a holding potential of −80 mV. Aslow,max decreased after applying either depolarizing or hyperpolarizing prepulses from −80 mV. The decline of Aslow,max after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3–S4 loop generated a Zn2+-binding site. At saturating concentrations, Aslow,max reached 100%, indicating that Zn2+ traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn2+ binding at −80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.

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