Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore dom... more Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between C...
A complete characterization of temperature -and voltage-activated TRP channel gating requires a p... more A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500 mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.
In this chapter the essentials of the kinetics of opening and closing of ionic channels will be d... more In this chapter the essentials of the kinetics of opening and closing of ionic channels will be discussed. I present several kinetic models and show the type of single channel records to be expected for each model and how to calculate kinetic constant from these records. The models I discuss are two state channels, three-state channels with three conductances, and three-state channels with only two conductances. The basic ideas for this chapter were taken from Dionne and Leibowitz (1982) and Ehrenstein et al. (1974).
The Jerusalem Symposia on Quantum Chemistry and Biochemistry, 1992
Molecular dynamics free energy perturbation simulations are used to examine the ion-binding energ... more Molecular dynamics free energy perturbation simulations are used to examine the ion-binding energetics and selectivity of the small ion-carrier valinomycin. Since available force fields have not been parametrised for the type of ion-carbonyl interactions encountered here, the results are used to determine carbonyl parameters that reproduce the observed selectivity quantitatively. These are then used in simulations of the energetics of alikaline-earth ion binding to the small Ca2+-binding protein calbindin D 9k to predict the selectivity of one site in this molecule. The dependence of selectivity on the dipole moment of the ion ligands is also examined and found to behave in accordance with early selectivity theories.
... 8. Binder, K.; Kalos, M. H. In Monte Carlo Methods In Staristical Physics; Binder, K., Ed.; S... more ... 8. Binder, K.; Kalos, M. H. In Monte Carlo Methods In Staristical Physics; Binder, K., Ed.; Springer-Verlag: New York, 1986; Chapter 6. (21) Butler, EP; Thomas, G. Acta Mer. ... SwedenOsvaldo Alvarez, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile ...
In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two... more In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two of the best characterized members of this class of channels: TRPM8 and TRPV1. Since channel activation by temperature is the hallmark of thermo-TRP channels, we present a detailed discussion on the thermodynamics involved in the gating processes by temperature, voltage, and agonists. We also review recently published data in an effort to put together all the pieces available of the amazing puzzle of thermo-TRP channel activation. Special emphasis is made in the structural components that allow the channel-forming proteins to integrate such diverse stimuli, and in the coupling between the different sensors and the ion conduction pathway. We conclude that the present data is most economically explained by allosteric models in which temperature, voltage, and agonists act separately to modulate channel activity.
The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permea... more The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K+ measured from biionic potentials were: NH4+ (1.52) greater than Rb+ (1.05) greater than K+ (1.0) greater than Cs+ (0.89) greater than Na+ (0.81) greater than Li+ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K+, Rb+, NH4+, Cs+, Na+ and Li+, respectively. Current-voltage curves for the different monovalent cations were measured and described using a three-barrier model previously used to explain the voltage dependence of the "instantaneous" channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH4+ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.
Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore dom... more Allosteric interplays between voltage-sensing domains (VSD), Ca2+-binding sites, and the pore domain govern the Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the Ca2+- and voltage-sensing mechanisms crosstalk on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation measuring and analyzing the effects of internal Ca2+ on BK channels gating currents. Our results indicate that the Ca2+ sensors occupancy has a strong impact on the VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, appear to contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between C...
A complete characterization of temperature -and voltage-activated TRP channel gating requires a p... more A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500 mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.
In this chapter the essentials of the kinetics of opening and closing of ionic channels will be d... more In this chapter the essentials of the kinetics of opening and closing of ionic channels will be discussed. I present several kinetic models and show the type of single channel records to be expected for each model and how to calculate kinetic constant from these records. The models I discuss are two state channels, three-state channels with three conductances, and three-state channels with only two conductances. The basic ideas for this chapter were taken from Dionne and Leibowitz (1982) and Ehrenstein et al. (1974).
The Jerusalem Symposia on Quantum Chemistry and Biochemistry, 1992
Molecular dynamics free energy perturbation simulations are used to examine the ion-binding energ... more Molecular dynamics free energy perturbation simulations are used to examine the ion-binding energetics and selectivity of the small ion-carrier valinomycin. Since available force fields have not been parametrised for the type of ion-carbonyl interactions encountered here, the results are used to determine carbonyl parameters that reproduce the observed selectivity quantitatively. These are then used in simulations of the energetics of alikaline-earth ion binding to the small Ca2+-binding protein calbindin D 9k to predict the selectivity of one site in this molecule. The dependence of selectivity on the dipole moment of the ion ligands is also examined and found to behave in accordance with early selectivity theories.
... 8. Binder, K.; Kalos, M. H. In Monte Carlo Methods In Staristical Physics; Binder, K., Ed.; S... more ... 8. Binder, K.; Kalos, M. H. In Monte Carlo Methods In Staristical Physics; Binder, K., Ed.; Springer-Verlag: New York, 1986; Chapter 6. (21) Butler, EP; Thomas, G. Acta Mer. ... SwedenOsvaldo Alvarez, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile ...
In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two... more In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two of the best characterized members of this class of channels: TRPM8 and TRPV1. Since channel activation by temperature is the hallmark of thermo-TRP channels, we present a detailed discussion on the thermodynamics involved in the gating processes by temperature, voltage, and agonists. We also review recently published data in an effort to put together all the pieces available of the amazing puzzle of thermo-TRP channel activation. Special emphasis is made in the structural components that allow the channel-forming proteins to integrate such diverse stimuli, and in the coupling between the different sensors and the ion conduction pathway. We conclude that the present data is most economically explained by allosteric models in which temperature, voltage, and agonists act separately to modulate channel activity.
The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permea... more The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K+ measured from biionic potentials were: NH4+ (1.52) greater than Rb+ (1.05) greater than K+ (1.0) greater than Cs+ (0.89) greater than Na+ (0.81) greater than Li+ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K+, Rb+, NH4+, Cs+, Na+ and Li+, respectively. Current-voltage curves for the different monovalent cations were measured and described using a three-barrier model previously used to explain the voltage dependence of the "instantaneous" channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH4+ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.
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