Hysteretic hERG Channel Gating Current Recorded At Physiological Temperature

Cardiac hERG channels comprise at least two subunits, hERG 1a and hERG 1b, and drive cardiac action potential repolarization. hERG 1a subunits contain a cytoplasmic PAS domain that is absent in hERG 1b. The hERG 1a PAS domain regulates voltage sensor domain (VSD) movement, but hERG VSD behavior and its regulation by the hERG 1a PAS domain have not been studied at physiological temperatures. We recorded gating charge from homomeric hERG 1a and heteromeric hERG 1a/1b channels at near physiological temperatures (36 ± 1°C) using pulse durations comparable in length to the human ventricular action potential. The voltage dependence of deactivation was hyperpolarized relative to activation, reflecting VSD relaxation at positive potentials. These data suggest that relaxation (hysteresis) works to delay pore closure during repolarization. Interestingly, hERG 1a VSD deactivation displayed a double Boltzmann distribution, but hERG 1a/1b deactivation displayed a single Boltzmann. Disabling the hERG1a PAS domain using a PAS-targeting antibody similarly transformed hERG 1a deactivation from a double to a single Boltzmann, highlighting the contribution of the PAS in regulating VSD movement. These data represent, to our knowledge, the first recordings of hERG gating charge at physiological temperature and demonstrate that VSD relaxation (hysteresis) is present in hERG channels at physiological temperature.

Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of −13.67 ± 9.02 (n = 7), −2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl−. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Maxwell Equations without a Polarization Field, Using a Paradigm from Biophysics

When forces are applied to matter, the distribution of mass changes. Similarly, when an electric field is applied to matter with charge, the distribution of charge changes. The change in the distribution of charge (when a local electric field is applied) might in general be called the induced charge. When the change in charge is simply related to the applied local electric field, the polarization field P is widely used to describe the induced charge. This approach does not allow electrical measurements (in themselves) to determine the structure of the polarization fields. Many polarization fields will produce the same electrical forces because only the divergence of polarization enters Maxwell’s first equation, relating charge and electric forces and field. The curl of any function can be added to a polarization field P without changing the electric field at all. The divergence of the curl is always zero. Additional information is needed to specify the curl and thus the structure of the P field. When the structure of charge changes substantially with the local electric field, the induced charge is a nonlinear and time dependent function of the field and P is not a useful framework to describe either the electrical or structural basis-induced charge. In the nonlinear, time dependent case, models must describe the charge distribution and how it varies as the field changes. One class of models has been used widely in biophysics to describe field dependent charge, i.e., the phenomenon of nonlinear time dependent induced charge, called ‘gating current’ in the biophysical literature. The operational definition of gating current has worked well in biophysics for fifty years, where it has been found to makes neurons respond sensitively to voltage. Theoretical estimates of polarization computed with this definition fit experimental data. I propose that the operational definition of gating current be used to define voltage and time dependent induced charge, although other definitions may be needed as well, for example if the induced charge is fundamentally current dependent. Gating currents involve substantial changes in structure and so need to be computed from a combination of electrodynamics and mechanics because everything charged interacts with everything charged as well as most things mechanical. It may be useful to separate the classical polarization field as a component of the total induced charge, as it is in biophysics. When nothing is known about polarization, it is necessary to use an approximate representation of polarization with a dielectric constant that is a single real positive number. This approximation allows important results in some cases, e.g., design of integrated circuits in silicon semiconductors, but can be seriously misleading in other cases, e.g., ionic solutions.

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.

Not so optimal: The evolution of mutual information in potassium voltage-gated channels

Potassium voltage-gated (Kv) channels need to detect and respond to rapidly changing ionic concentrations in their environment. With an essential role in regulating electric signaling, they would be expected to be optimal sensors that evolved to predict the ionic concentrations. To explore these assumptions, we use statistical mechanics in conjunction with information theory to model how animal Kv channels respond to changes in potassium concentrations in their environment. By estimating mutual information in representative Kv channel types across a variety of environments, we find two things. First, under a wide variety of environments, there is an optimal gating current that maximizes mutual information between the sensor and the environment. Second, as Kv channels evolved, they have moved towards decreasing mutual information with the environment. This either suggests that Kv channels do not need to act as sensors of their environment or that Kv channels have other functionalities that interfere with their role as sensors of their environment.

Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

Quantum calculations on 976 atoms of the voltage sensing domain of the K_v1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H⁺transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H⁺ transfer, not motion of arginine charges.

Quantum Calculation of Proton and Other Charge Transfer Steps in Voltage Sensing in the Kv1.2 Channel

Quantum calculations on 976 atoms of the voltage sensing domain of the K_v1.2 channel, with protons in several positions, give energy, charge transfer, and other properties. Motion of the S4 transmembrane segment that accounts for gating current in standard models is shown not to occur; there is H⁺transfer instead. The potential at which two proton positions cross in energy approximately corresponds to the gating potential for the channel. The charge displacement seems approximately correct for the gating current. Two mutations are accounted for (Y266F, R300cit, cit =citrulline). The primary conclusion is that voltage sensing depends on H⁺ transfer, not motion of arginine charges.

The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations

Over two-thirds of a century ago, Hodgkin and Huxley proposed the existence of voltage gated ion channels (VGICs) to carry Na+ and K+ ions across the cell membrane to create the nerve impulse, in response to depolarization of the membrane. The channels have multiple physiological roles, and play a central role in a wide variety of diseases when they malfunction. The first channel structure was found by MacKinnon and coworkers in 1998. Subsequently, the structure of a number of VGICs was determined in the open (ion conducting) state. This type of channel consists of four voltage sensing domains (VSDs), each formed from four transmembrane (TM) segments, plus a pore domain through which ions move. Understanding the gating mechanism (how the channel opens and closes) requires structures. One TM segment (S4) has an arginine in every third position, with one such segment per domain. It is usually assumed that these arginines are all ionized, and in the resting state are held toward the intracellular side of the membrane by voltage across the membrane. They are assumed to move outward (extracellular direction) when released by depolarization of this voltage, producing a capacitive gating current and opening the channel. We suggest alternate interpretations of the evidence that led to these models. Measured gating current is the total charge displacement of all atoms in the VSD; we propose that the prime, but not sole, contributor is proton motion, not displacement of the charges on the arginines of S4. It is known that the VSD can conduct protons. Quantum calculations on the Kv1.2 potassium channel VSD show how; the key is the amphoteric nature of the arginine side chain, which allows it to transfer a proton. This appears to be the first time the arginine side chain has had its amphoteric character considered. We have calculated one such proton transfer in detail: this proton starts from a tyrosine that can ionize, transferring to the NE of the third arginine on S4; that arginine’s NH then transfers a proton to a glutamate. The backbone remains static. A mutation predicted to affect the proton transfer has been qualitatively confirmed experimentally, from the change in the gating current-voltage curve. The total charge displacement in going from a normal closed potential of −70 mV across the membrane to 0 mV (open), is calculated to be approximately consistent with measured values, although the error limits on the calculation require caution in interpretation.