Patch electrode glass composition affects ion channel currents.

The influence of patch electrode glass composition on macroscopic IV relations in inside-out patches of the cGMP-activated ion channel from rod photoreceptors was examined for a soda lime glass, a Kovar sealing glass, a borosilicate glass, and several soft lead glasses. In several glasses the shape or magnitude of the currents changed as the concentration of EGTA or EDTA was increased from 200 microM to 10 mM. The changes in IV response suggest that, at low concentrations of chelator, divalent cations are released from the electrode glass and interact with the cGMP-activated channel. Possible mechanisms are discussed to explain the observations, and several comments are made concerning the choice of a glass for patching.     

Ligand-gated ion channel currents in a nonstationary lyotropic model

Transmembrane currents in ligand-gated ion channels are calculated in a nonstationary, chemically open whole cell system or patch of a membrane. The model is lyotropic in the sense that dynamics, and parameters such as the ligand concentration for half-maximal response (scale of response), and threshold for firing, such as in neurons, become nonlinear functions of the reactant concentrations. The derived total currents fit recorded data significantly better than those derived from mass action, Ising, and other stationary type models, in which the derived response is often displaced from the assessed response by several orders in the ligand concentration. Also, the derived slope of response is in perfect agreement with the values assessed.     

Estimation of ion channel kinetics from fluctuations of macroscopic currents

For single channel recordings, the maximum likelihood estimation (MLE) of kinetic rates and conductance is well established. A direct extrapolation of this method to macroscopic currents is computationally prohibitive: it scales as a power of the number of channels. An approximated MLE that ignored the local time correlation of the data has been shown to provide estimates of the kinetic parameters. In this article, an improved approximated MLE that takes into account the local time correlation is proposed. This method estimates the channel kinetics using both the time course and the random fluctuations of the macroscopic current generated by a homogeneous population of ion channels under white noise. It allows arbitrary kinetic models and stimulation protocols. The application of the proposed algorithm to simulated data from a simple three-state model on nonstationary conditions showed reliable estimates of all the kinetic constants, the conductance and the number of channels, and reliable values for the standard error of those estimates. Compared to the previous approximated MLE, it reduces by a factor of 10 the amount of data needed to secure a given accuracy and it can even determine the kinetic rates in macroscopic stationary conditions.     

Maximum likelihood estimation of ion channel kinetics from macroscopic currents

We describe a maximum likelihood method for direct estimation of rate constants from macroscopic ion channel data for kinetic models of arbitrary size and topology. The number of channels in the preparation, and the mean and standard deviation of the unitary current can be estimated, and a priori constraints can be imposed on rate constants. The method allows for arbitrary stimulation protocols, including stimuli with finite rise time, trains of ligand or voltage steps, and global fitting across different experimental conditions. The initial state occupancies can be optimized from the fit kinetics. Utilizing arbitrary stimulation protocols and using the mean and the variance of the current reduce or eliminate problems of model identifiability (Kienker, 1989). The algorithm is faster than a recent method that uses the full autocovariance matrix (Celentano and Hawkes, 2004), in part due to the analytical calculation of the likelihood gradients. We tested the method with simulated data and with real macroscopic currents from acetylcholine receptors, elicited in response to brief pulses of carbachol. Given appropriate stimulation protocols, our method chose a reasonable model size and topology.     

Phase transitions and ion currents in a model ferroelectric channel unit

The hypothesis of ferroelectric electrodiffusion is examined mathematically. A thermodynamic potential, the elastic Gibbs function, written in polynomial form, provides the dielectric equation of state for the model. The other equations of electrodiffusion theory complete the model. This system reduces to a second-order partial differential equation, which is formally solved by the phase-plane method. This solution, applied to the Na channel, leads to a propagating phase-transition wave accompanied by movement of ionic charge. This may be readily interpreted as a transmembrane wave traveling along a ferroelectric unit within, and transporting ions through, the channel. Comparison of the temperature dependence of axonal conduction velocity with that of the spontaneous polarization of Rochelle salt suggests that the Na channel of squid axon contains a ferroelectric unit having a lower Curie point, but decomposing before reaching its upper Curie point. Comparison with data from reconstitution experiments suggests that the ferroelectric unit is a carbohydrate enclosed in an intrinsic protein structure to form a glycoprotein channel. The value experimentally estimated for the surface charge of the Na channel is within the range of spontaneous polarizations of typical ferroelectric crystals. It is argued that the ferroelectric probably is a single crystal of the order-disorder type, which undergoes a first-order transition between a ferroelectric and a paraelectric state during excitational activity. The hypothesis of ferroelectric channel units is consistent with the existence and directionality of the observed "gating" currents.     

Frequency response of alternating currents through the Staphylococcus aureus alpha-hemolysin ion channel

Alternating currents were measured through transmembrane ion channels formed by Staphylococcus aureus alpha-hemolysin proteins in planar bilayer membranes as part of an investigation to determine the channel's frequency response and the appropriateness of an equivalent circuit commonly used to model electrical interactions at the surface of cells. The experimental approach includes a novel method for separating the alternating current through one or more channels, which is conductive in nature, from the capacitively coupled current through the membrane. Separation of the conductive and capacitive alternating currents made it possible to measure the frequency response of the alpha-hemolysin channels. The results of the study are consistent with an equivalent circuit of a membrane capacitor in parallel with one or more channel resistors over the frequency range 30-120 Hz. The possible usefulness of frequency response data for ion channels in cell membranes during investigations of biological effects of time-varying magnetic fields is briefly discussed.     

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel

Summary A study of the temperature dependence of gramicidin A conductance of K⁺ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.     

Quantitative modeling of currents from a voltage gated ion channel undergoing fast inactivation

Ion channels play a central role in setting gradients of ion concentration and electrostatic potentials, which in turn regulate sensory systems and other functions. Based on the structure of the open configuration of the Kv1.2 channel and the suggestion that the two ends of the N-terminal inactivating peptide form a bivalent complex that simultaneously blocks the channel pore and binds to the cytoplasmic T1 domain, we propose a six state kinetic model that for the first time reproduces the kinetics of recovery of the Drosophila Shaker over the full range of time scales and hyperpolarization potentials, including tail currents. The model is motivated by a normal mode analysis of the inactivated channel that suggests that a displacement consistent with models of the closed state propagates to the T1 domain via the S1-T1 linker. This motion stretches the bound (inactivating) peptide, hastening the unblocking of the pore. This pulling force is incorporated into the rates of the open to blocked states, capturing the fast recovery phase of the current for repolarization events shorter than 1 ms. If the membrane potential is hyperpolarized, essential dynamics further suggests that the T1 domain returns to a configuration where the peptide is un-stretched and the S1-T1 linker is extended. Coupling this novel hyperpolarized substate to the closed, open and blocked pore states is enough to quantitatively estimate the number of open channels as a function of time and membrane potential. A straightforward prediction of the model is that a slow ramping of the potential leads to very small currents.     

Fluctuations in ion channel gating currents. Analysis of nonstationary shot noise.

Conti and Stühmer (1989. Eur. Biophys. J. 17:53-59) have measured the nonstationary shot noise in the gating current of a population of sodium channels. Here we present expressions for the autocovariance and variance of such noise from general Markov models of channel kinetics, based on the theoretical work of E. Frehland. We compare the predictions of the independent, two-state gating model used by Conti and Stühmer with a six-state model of sodium channel activation based on the work of Armstrong and Gilly (1979. J. Gen. Physiol. 74:691-711). We find that Conti and Stühmer's experiment would not be able to distinguish between these schemes. We describe experimental conditions under which better model discrimination would be possible.     

Estimating rate constants from single ion channel currents when the initial distribution is known

Single ion channel currents can be analysed by hidden or aggregated Markov models. A classical result from Fredkin et al. (Proceedings of the Berkeley conference in honor of Jerzy Neyman and Jack Kiefer, vol I, pp 269–289, 1985) states that the maximum number of identifiable parameters is bounded by 2n_on_c, where n_o and n_c denote the number of open and closed states, respectively. We show that this bound can be overcome when the probabilities of the initial distribution are known and the data consist of several sweeps.     

Expressing acid-sensing ion channel 3 in the brain alters acid-evoked currents and impairs fear conditioning

Previous studies on mice with a disruption of the gene encoding acid-sensing ion channel 1a (ASIC1a) suggest that ASIC1a is required for normal fear behavior. To investigate the effects of altering the subunit composition of brain ASICs on behavior, we developed transgenic mice expressing ASIC3 via the pan-neuronal synapsin I promoter. These mice express ASIC3 in the brain, where the endogenous ASIC3 protein is not detected. We found that in ASIC3 transgenic mice, ASIC3 co-immunoprecipitated with the endogenous ASIC1a protein and distributed in the same subcellular brain fractions as ASIC1a. In addition, ASIC3 significantly increased the rate of desensitization of acid-evoked currents in cultured cortical neurons. Importantly, ASIC3 reduced Pavlovian fear conditioning to both context and auditory cues. These observations suggest that ASIC3 can heteromultimerize with ASIC1a in the brain and alter the biophysical properties of the endogenous channel complex. Moreover, these data suggest that ASIC subunit composition and channel desensitization may be critical determinants for ASIC-dependent behavior.     

High-throughput screening for ion channel modulators

Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.     

Evaluation of agonist selectivity for the NMDA receptor ion channel in bilayer lipid membranes based on integrated single-channel currents

A new method for evaluating chemical selectivity of agonists to activate the N-methyl-D-aspartate (NMDA) receptor was presented by using typical agonists NMDA, L-glutamate and (2S, 3R, 4S)-2-(carboxycyclopropyl)glycine (L-CCG-IV) and the mouse epsilon1/zeta1 NMDA receptor incorporated in bilayer lipid membranes (BLMs) as an illustrative example. The method was based on the magnitude of an agonist-induced integrated single-channel current corresponding to the number of total ions passed through the open channel. The very magnitudes of the integrated single-channel currents were compared with the different BLMs as a new measure of agonist selectivity. The epsilon1/zeta1 NMDA receptor was partially purified from Chinese hamster ovary (CHO) cells expressing the epsilon1/zeta1 NMDA receptor and incorporated in BLMs formed by the tip-dip method. The agonist-induced integrated single-channel currents were obtained at 50 microM agonist concentration, where the integrated current for NMDA was shown to reach its saturated value. The obtained integrated currents were found to be (4.5 +/- 0.55) x 10(-13) C/s for NMDA, (5.8 +/- 0.72) x 10(-13) C/s for L-glutamate and (6.6 +/- 0.61) x 10(-13) C/s for L-CCG-IV, respectively. These results suggest that the agonist selectivity in terms of the total ion flux through the single epsilon1/zeta1 NMDA receptor is in the order of L-CCG-IV approximately = L-glutamate > NMDA.     

Group II metabotropic glutamate receptor activation attenuates acid-sensing ion channel currents in rat primary sensory neurons

Acid-sensing ion channels (ASICs) play an important role in pain associated with tissue acidification. Peripheral inhibitory group II metabotropic glutamate receptors (mGluRs) have analgesic effects in a variety of pain conditions. Whether there is a link between ASICs and mGluRs in pain processes is still unclear. Herein, we show that the group II mGluR agonist {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 inhibited acid-evoked ASIC currents and action potentials in rat dorsal root ganglia neurons. {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 reduced the maximum current response to protons, but it did not change the sensitivity of ASICs to protons. {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 inhibited ASIC currents by activating group II mGluRs. We found that the inhibitory effect of {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 was blocked by intracellular application of the G_i/o protein inhibitor pertussis toxin and the cAMP analogue 8-Br-cAMP and mimicked by the protein kinase A (PKA) inhibitor H-89. {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 also inhibited ASIC3 currents in CHO cells coexpressing mGluR2 and ASIC3 but not in cells expressing ASIC3 alone. In addition, intraplantar injection of {"type":"entrez-nucleotide","attrs":{"text":"LY354740","term_id":"1257481336","term_text":"LY354740"}}LY354740 dose-dependently alleviated acid-induced nociceptive behavior in rats through local group II mGluRs. Together, these results suggested that activation of peripheral group II mGluRs inhibited the functional activity of ASICs through a mechanism that depended on G_i/o proteins and the intracellular cAMP/PKA signaling pathway in rat dorsal root ganglia neurons. We propose that peripheral group II mGluRs are an important therapeutic target for ASIC-mediated pain.     

Model channel ion currents in NaCl-extended simple point charge water solution with applied-field molecular dynamics.

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125-A internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and extended simple point charge water. Channel and bath currents were computed from 10 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration dependent and superlinear at high concentrations.     

Biphasic currents evoked by chemical or thermal activation of the heat-gated ion channel, TRPV3

2-Aminoethyl diphenylborinate was recently identified as a chemical activator of TRPV1, TRPV2, and TRPV3, three heat-gated members of the transient receptor potential vanilloid (TRPV) ion channel subfamily. Here we demonstrated that two structurally related compounds, diphenylboronic anhydride (DPBA) and diphenyltetrahydrofuran (DPTHF), can also modulate the activity of these channels. DPBA acted as a TRPV3 agonist, whereas DPTHF exhibited prominent antagonistic activity. However, all three diphenyl-containing compounds promoted some degree of channel activation or potentiation, followed by channel block. Strong TRPV3 activation by DPBA often leads to the appearance of a secondary, enhanced, current phase. A similar biphasic response was observed during TRPV3 heat stimulation; an initial, gradually sensitizing phase (I(1)) was followed by an abrupt transition to a secondary phase (I(2)). I(2) was characterized by larger current amplitude, loss of outward rectification, and alterations in the following properties: permeability among cations; ruthenium red and DPTHF sensitivity; temperature dependence; and voltage-dependent gating. The I(1) to I(2) transition depended strongly on TRPV3 current density. Removal of extracellular divalent cations resulted in heat-evoked currents resembling I(2), whereas mutation of a putative Ca(2+)-binding residue in the pore loop domain, aspartate 641, facilitated detection of the I(1) to I(2) transition, suggesting that the conversion to I(2) resulted from the agonist- and time-dependent loss of divalent cationic inhibition. Primary keratinocytes overexpressing exogenous TRPV3 also exhibited biphasic agonist-evoked currents. Thus, strong activation by either chemical or thermal stimuli led to biphasic TRPV3 signaling behavior that may be associated with changes in the channel pore.     

Mechanosensitivity of N-type calcium channel currents

Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.     

Single channel currents in adrenocortical cells

Single channel currents have been recorded from cell-attached patches of tumoral adrenocortical cells. Our experiments suggest the existence of three sets of potassium channels in the surface membrane of these cells. All channel types can be recorded in a given membrane patch but some patches have only one type of single channel currents. One channel type has a unitary conductance of about 103 pS. The other two channels have smaller conductances and opposite voltage dependence. In one case channels open on depolarization and have a single channel conductance of 31.6 pS. In the other case the probability of being in the open state increases on hyperpolarization and the single channel conductance is of 21 pS. These channels seem to be similar to the delayed and anomalous rectifying potassium channels seen in other preparations. The role of membrane ionic permeability in steroid release induced by ACTH is discussed.