Direct cardiotoxicity of lead

Lead is a heavy metal pollutant that constitutes frequent exposomes. It is nonbiodegradable and has a nonsafe limit of exposure. It has multisystemic effects, and most of the cardiac effects have been discovered to be indirect. There are strong similarities between Ca2+ and Pb2+ in their chemistry. Because cardiac function is dramatically dependent in extracellular Ca2+, as well as in precise control of intracellular Ca2+, we tested if Pb2+ could antagonize Ca2+-dependent effects in a short amount of time. Acute exposure of isolated hearts showed a negative inotropic effect. In guinea pig isolated cardiomyocytes loaded with a Pb2+-specific dye (Leadmium green), our results showed that there was an associated increment in fluorescence related to extracellular stimulation blocked by 1–5 µM DHP. Calcium currents were partially blocked by extracellular Pb2+, though currents seemed to last longer after a fast inactivation. Charge movement from gating currents was slightly hastened over time, giving an appearance of a slight reduction in the Cav1.2 gating currents. Action potentials were prolonged in Pb2+ compared with Ca2+. In isolated cardiomyocytes loaded with Ca2+-sensitive dyes, Ca2+ variations promoted by extracellular stimuli were affected in space/time. As Pb2+ could interfere with Ca2+-sensitive dyes, we measured contraction of isolated cardiomyocytes under extracellular stimuli in Pb2+. In both Ca2+ dye fluorescence and contractions, Pb2+ disorganizes the pattern of contraction and intracellular Ca2+ homeostasis. Our results suggest that (1) Pb2+ enters to cardiomyocytes through Cav1.2 channels, and (2) once it enters the cell, Pb2+ may substitute Ca2+ in Ca2+-binding proteins. In addition to these direct mechanisms related to Pb2+ competition with Ca2+-binding sites, we cannot discard a direct contribution of Pb2+ redox properties.

Snapin Specifically Up-Regulates Cav1.3 Ca2+ Channel Variant with a Long Carboxyl Terminus

Ca2+ entry through Cav1.3 Ca2+ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Cav1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CTL) of rat Cav1.3L variant. Co-expression of Snapin with Cav1.3L/Cavβ3/α2δ2 subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Cav1.3S containing a short CT (CTS) in which a Snapin interaction site in the CTL was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Cav1.3L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Cav1.3L. Taken together, our results strongly suggest that Snapin directly interacts with the CTL of Cav1.3L, leading to up-regulation of Cav1.3L channel activity via facilitating channel opening probability.

The voltage sensor is responsible for ΔpH dependence in Hv1 channels

The dissipation of acute acid loads by the voltage-gated proton channel (Hv1) relies on regulating the channel’s open probability by the voltage and the ΔpH across the membrane (ΔpH = pHex − pHin). Using monomeric Ciona-Hv1, we asked whether ΔpH-dependent gating is produced during the voltage sensor activation or permeation pathway opening. A leftward shift of the conductance-voltage (G-V) curve was produced at higher ΔpH values in the monomeric channel. Next, we measured the voltage sensor pH dependence in the absence of a functional permeation pathway by recording gating currents in the monomeric nonconducting D160N mutant. Increasing the ΔpH leftward shifted the gating charge-voltage (Q-V) curve, demonstrating that the ΔpH-dependent gating in Hv1 arises by modulating its voltage sensor. We fitted our data to a model that explicitly supposes the Hv1 voltage sensor free energy is a function of both the proton chemical and the electrical potential. The parameters obtained showed that around 60% of the free energy stored in the ΔpH is coupled to the Hv1 voltage sensor activation. Our results suggest that the molecular mechanism underlying the Hv1 ΔpH dependence is produced by protons, which alter the free-energy landscape around the voltage sensor domain. We propose that this alteration is produced by accessibility changes of the protons in the Hv1 voltage sensor during activation.

The Joy of Markov Models—Channel Gating and Transport Cycling Made Easy

ABSTRACT The behavior of ion channels and transporters is often modeled using discrete state continuous-time Markov models. Such models are helpful for the interpretation of experimental data and can guide the design of experiments by testing specific predictions. Here, we describe a computational tool that allows us to create Markov models of chosen complexity and to calculate the predictions on a macroscopic scale, as well on a single-molecule scale. The program calculates steady-state properties (current, state probabilities, and cycle frequencies), deterministic macroscopic and stochastic time courses, gating currents, dwell-time histograms, and power spectra of channels and transporters. In addition, a visual simulation mode allows us to follow the time-dependent stochastic behavior of a single channel or transporter. After a basic introduction into the concept of Markov models, real-life examples are discussed, including a model of a simple K+ channel, a voltage-gated sodium channel, a 3-state ligand-gated channel, and an electrogenic uniporter. In this manner, the article has a modular architecture, progressing from basic to more advanced topics. This illustrates how the MarkovEditor program can serve students to explore Markov models at a basic level but is also suited for research scientists to test and develop models on the mechanisms of protein function.

Gating current noise produced by Brownian models of a voltage sensor

The activation of voltage-dependent ion channels is associated with the movement gating charges, that give rise to gating currents. Although gating currents originating from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents originating from a population of channels can make a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a Markov model of channel activation, are in accordance with the presence of a main step along the activation pathway carrying 2.3-2.4 e0 of charge. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we employed a Brownian model of voltage-dependent gating that we recently developed using structural information and applying the laws of electrodynamics. The model was capable to reproduce gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of this model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real ion channels the voltage sensor does not move in a fully Markovian regimen due to the relatively low (<5 kT) energy barriers separating successive intermediate states. As a consequence, the simultaneous jump of multiple gating charges through the gating pore becomes frequent, and this occurrence is at the origin of the relatively high single-step charge detected by assuming Markovian behavior.

Broadband Tuning the Voltage Dependence of a Sodium Channel

ABSTRACTVoltage-gated sodium channels initiate action potentials in prokaryotes and in many eukaryotic cells, including vertebrate nerve and muscle. Their activation is steeply voltage-dependent, but it is unclear how the voltage sensitivity is set or whether it can be broadly shifted to positive voltages. Here we show that the voltage dependence of activation (VA) of the ancestral bacterial sodium channel NaVAb can be progressively shifted from −118 mV to +35 mV in chimeras with increasing numbers of amino acid residues from the extracellular half of the voltage sensor of human NaV1.7 channels. In a minimal chimera in which only 32 residues were transferred, we analyzed the effects of six additional mutations of conserved amino acid residues singly, in pairs, and as triple mutations. The resulting chimeric mutants exhibited a broad range of voltage sensitivity from VA=−118 mV to VA=+120 mV. Three mutations (N48K, L112A, and M119V) shifted VA to +61 mV when substituted in NaVAb itself, and substitution of two additional Cys residues in the Cys-free background of NaVAb further shifted VA to +105 mV. In these mutants, measurement of gating currents revealed that the voltage dependence of gating charge movement (VQ) shifted to positive membrane potentials as much or more than VA, confirming that the gating charges are trapped in their resting positions by these VA-shifting mutations. Our results demonstrate broadband shifting of VA and VQ of a sodium channel across a range of 240 mV and provide a toolbox of methods and constructs to analyze sodium channel structure and function in the resting state at 0 mV and in activated states at positive membrane potentials.GRAPHICAL ABSTRACTThe complete range of broadband tuning of voltage-dependent activation of a sodium channel.

Coupling of Ca2+and voltage activation in BK channels through the αB helix/voltage sensor interface

Large-conductance Ca2+and voltage-activated K+(BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+activation for both Ca2+bowl and RCK1 Ca2+sites, suggesting that both high-affinity Ca2+sites transduce their effect, at least in part, through the αB helix. Mg2+activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+binding and voltage depolarization to pore opening and that shared Ca2+and voltage transduction pathways involving the αB helix may be involved.

Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface

Large conductance Ca2+ and voltage activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is comprised of a voltage sensor domain (VSD), a central pore gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We now explore mechanism by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the HCA model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation was also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to WT, without other notable differences in the CTD, indicating structural change from the mutation was confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening, and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.SignificanceLarge conductance BK (Slo1) K+ channels are activated by voltage, Ca2+, and Mg2+ to modulate membrane excitability in neurons, muscle, and other cells. BK channels are of modular design, with pore-gate and voltage sensors as transmembrane domains and a large cytoplasmic domain CTD containing the Ca2+ sensors. Previous observations suggest that voltage and Ca2+ sensors interact, but less is known about this interaction and its involvement in the gating process. We show that a previously identified structural interface between the CTD and voltage sensors is required for effective activation by both voltage and Ca2+, suggesting that these processes may share common allosteric activation pathways. Such knowledge should help explain disease processes associated with BK channel dysfunction.

Calcium-driven regulation of voltage-sensing domains in BK channels

Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on 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, 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 Ca2+ sensors and VSDs.