scholarly journals Detection of the Opening of the Bundle Crossing in KcsA with Fluorescence Lifetime Spectroscopy Reveals the Existence of Two Gates for Ion Conduction

2006 ◽  
Vol 128 (5) ◽  
pp. 569-581 ◽  
Author(s):  
Rikard Blunck ◽  
Julio F. Cordero-Morales ◽  
Luis G. Cuello ◽  
Eduardo Perozo ◽  
Francisco Bezanilla
Keyword(s):  
Steady State ◽  
Fluorescence Lifetime ◽  
Channel Structure ◽  
Relative Distribution ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Ion Conduction ◽  
Ion Flow ◽  
Channel Opening ◽  
Direct Recording

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.

scholarly journals Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

2014 ◽  
Vol 143 (2) ◽  
pp. 289-307 ◽  
Author(s):  
Line Garneau ◽  
Hélène Klein ◽  
Marie-France Lavoie ◽  
Emmanuelle Brochiero ◽  
Lucie Parent ◽  
...  
Keyword(s):  
Selectivity Filter ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Channel Pore ◽  
Transmembrane Segments ◽  
Aromatic Interactions ◽  
C Terminus ◽  
Helical Segment ◽  
Channel Opening ◽  
Dynamics Simulations

The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators.

scholarly journals An S6 Mutation in BK Channels Reveals β1 Subunit Effects on Intrinsic and Voltage-dependent Gating

2006 ◽  
Vol 128 (6) ◽  
pp. 731-744 ◽  
Author(s):  
Bin Wang ◽  
Robert Brenner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Α Subunit ◽  
Channel Opening ◽  
Sensor Activation ◽  
Intracellular Domains

Large conductance, Ca2+- and voltage-activated K+ (BK) channels are exquisitely regulated to suit their diverse roles in a large variety of physiological processes. BK channels are composed of pore-forming α subunits and a family of tissue-specific accessory β subunits. The smooth muscle–specific β1 subunit has an essential role in regulating smooth muscle contraction and modulates BK channel steady-state open probability and gating kinetics. Effects of β1 on channel's gating energetics are not completely understood. One of the difficulties is that it has not yet been possible to measure the effects of β1 on channel's intrinsic closed-to-open transition (in the absence of voltage sensor activation and Ca2+ binding) due to the very low open probability in the presence of β1. In this study, we used a mutation of the α subunit (F315Y) that increases channel openings by greater than four orders of magnitude to directly compare channels' intrinsic open probabilities in the presence and absence of the β1 subunit. Effects of β1 on steady-state open probabilities of both wild-type α and the F315Y mutation were analyzed using the dual allosteric HA model. We found that mouse β1 has two major effects on channel's gating energetics. β1 reduces the intrinsic closed-to-open equilibrium that underlies the inhibition of BK channel opening seen in submicromolar Ca2+. Further, PO measurements at limiting slope allow us to infer that β1 shifts open channel voltage sensor activation to negative membrane potentials, which contributes to enhanced channel opening seen at micromolar Ca2+ concentrations. Using the F315Y α subunit with deletion mutants of β1, we also demonstrate that the small N- and C-terminal intracellular domains of β1 play important roles in altering channel's intrinsic opening and voltage sensor activation. In summary, these results demonstrate that β1 has distinct effects on BK channel intrinsic gating and voltage sensor activation that can be functionally uncoupled by mutations in the intracellular domains.

scholarly journals Coupling between Voltage Sensor Activation, Ca2+ Binding and Channel Opening in Large Conductance (BK) Potassium Channels

2002 ◽  
Vol 120 (3) ◽  
pp. 267-305 ◽  
Author(s):  
Frank T. Horrigan ◽  
Richard W. Aldrich
Keyword(s):  
Steady State ◽  
Conformational Change ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Kinetic Properties ◽  
Open Probability ◽  
Voltage Sensors ◽  
Channel Opening ◽  
Wide Range ◽  
Sensor Activation

To determine how intracellular Ca2+ and membrane voltage regulate the gating of large conductance Ca2+-activated K+ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca2+ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca2+, the steady-state gating charge-voltage (QSS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (GK-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca2+. This change reflects a differential effect of Ca2+ on voltage sensor activation and channel opening. Ca2+ has only a small effect on the fast component of ON gating current, indicating that Ca2+ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca2+, demonstrating that Ca2+ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca2+ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca2+ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca2+ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic IK kinetics indicate that Ca2+ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.

scholarly journals Irreversible temperature gating in trpv1 sheds light on channel activation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ana Sánchez-Moreno ◽  
Eduardo Guevara-Hernández ◽  
Ricardo Contreras-Cervera ◽  
Gisela Rangel-Yescas ◽  
Ernesto Ladrón-de-Guevara ◽  
...  
Keyword(s):  
Trp Channels ◽  
Structural Information ◽  
Heat Absorption ◽  
Absorption Process ◽  
Open Probability ◽  
Temperature Changes ◽  
Trpv1 Channels ◽  
Channel Opening ◽  
Temperature Activation ◽  
Thermal Stimuli

Temperature-activated TRP channels or thermoTRPs are among the only proteins that can directly convert temperature changes into changes in channel open probability. In spite of a wealth of functional and structural information, the mechanism of temperature activation remains unknown. We have carefully characterized the repeated activation of TRPV1 by thermal stimuli and discovered a previously unknown inactivation process, which is irreversible. We propose that this form of gating in TRPV1 channels is a consequence of the heat absorption process that leads to channel opening.

scholarly journals Activation of Ca2+-activated K+(maxi-K+) channel by angiotensin II in myocytes of the guinea pig ileum

1998 ◽  
Vol 274 (4) ◽  
pp. C983-C991 ◽  
Author(s):  
Fernando Romero ◽  
Bagnólia A. Silva ◽  
Viviane L. A. Nouailhetas ◽  
Jeannine Aboulafia
Keyword(s):  
Angiotensin Ii ◽  
K Channel ◽  
Intermediate Step ◽  
Synthetic Analog ◽  
Ang Ii ◽  
Open Probability ◽  
Channel Opening ◽  
Intracellular Ca ◽  
Patch Configuration ◽  
Pharmacomechanical Coupling

We investigated the regulation of the Ca2+-activated K+(maxi-K+) channel by angiotensin II (ANG II) and its synthetic analog, [Lys2]ANG II, in freshly dispersed intestinal myocytes. We identified a maxi-K+ channel population in the inside-out patch configuration on the basis of its conductance (257 ± 4 pS in symmetrical 150 mM KCl solution), voltage and Ca2+ dependence of channel opening, low Na+-to-K+and Cl−-to-K+permeability ratios, and blockade by external Cs+ and tetraethylammonium chloride. ANG II and [Lys2]ANG II caused an indirect, reversible, Ca2+- and dose-dependent activation of maxi-K+ channels in cell-attached experiments when cells were bathed in high-K+ solution. This effect was reversibly blocked by DUP-753, being that it is mediated by the AT1 receptor. Evidences that activation of the maxi-K+ channel by ANG II requires a rise in intracellular Ca2+concentration ([Ca2+]i) as an intermediate step were the shift of the open probability of the channel-membrane potential relationship to less positive membrane potentials and the sustained increase in [Ca2+]iin fura 2-loaded myocytes. The preservation of the pharmacomechanical coupling of ANG II in these cells provides a good model for the study of transmembrane signaling responses to ANG II and analogs in this tissue.

Phase-Resolved Fluorescence Spectral and Lifetime Characterization of Commercial Humic Substances

1997 ◽  
Vol 51 (7) ◽  
pp. 921-929 ◽  
Author(s):  
Sherry L. Hemmingsen ◽  
Linda B. McGown
Keyword(s):  
Steady State ◽  
Humic Substances ◽  
Direct Measurement ◽  
Fluorescence Lifetime ◽  
Spectral Features ◽  
Good Precision ◽  
Convenient Means ◽  
Accurate Comparison ◽  
Excitation Emission Matrices

Phase-resolved excitation-emission matrices (PREEMs) are shown to provide a unique visual representation of the intrinsic fluorescence properties of humic acids under a variety of solution conditions. The calculation of spectral peak ratios in PREEMs as well as steady-state excitation-emission matrices provides a convenient means for quantitating differences between the spectra with good precision. Absorbance correction is shown to be essential for accurate comparison among spectral features. Increased detail is available from PREEMs at various modulation frequencies that reveal the distribution of fluorescence lifetime contributions across the spectral surface. Direct measurement of fluorescence lifetime recovered three ranges of lifetime components in the humic substances, <1 ns, 2–5 ns, and 8–14 ns, that are consistent with previously reported lifetimes. PREEMs, which provide a concise “survey” of how the lifetimes change across the spectrum, may aid in pinpointing spectral regions that provide the best lifetime discrimination among samples.

scholarly journals Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).

1992 ◽  
Vol 99 (6) ◽  
pp. 841-862 ◽  
Author(s):  
F Markwardt ◽  
G Isenberg
Keyword(s):  
Steady State ◽  
Time Course ◽  
Boltzmann Distribution ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
K Channels ◽  
The Mean ◽  
Inside Out

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.

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