Comparing ion conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels

In this article we compare electrical conductance events from single channel recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in human embryonic kidney cells with channel events recorded on synthetic lipid membranes close to melting transitions. Ion channels from the TRP family are involved in a variety of sensory processes including thermo- and mechano-reception. Synthetic lipid membranes close to phase transitions display channel-like events that respond to stimuli related to changes in intensive thermodynamic variables such as pressure and temperature. TRP channel activity is characterized by typical patterns of current events dependent on the type of protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical phenomena that are considered typical for the activity of protein ion channels. We find unitary currents, burst behavior, flickering, multistep-conductances, and spikes behavior in both preparations. Moreover, we report conductances and lifetimes for lipid channels as described for protein channels. Non-linear and asymmetric current-voltage relationships are seen in both systems. Without further knowledge of the recording conditions, no easy decision can be made whether short current traces originate from a channel protein or from a pure lipid membraneComment: 13 pages, 9 Figure

Sodium channel inactivation kinetics of rat sensory and motor nerve fibres and their modulation by glutathione

Na+ channel currents of rat motor and sensory nerve fibres were studied with the patch-clamp technique on enzymatically demyelinated axons. Differences between motor and sensory fibres in multi-channel inactivation kinetics and the gating of late single-channel currents were investigated. In the axon-attached mode, inactivation of multi-channel Na+ currents in sensory axons was best fitted with a single time constant while for motor axons two time constants were needed. Late single-channel currents in sensory axons were characterized by short openings whereas motor axons exhibited additional long single-channel openings. In contrast, in excised, inside-out membrane patches, no differences between motor and sensory fibres were found: in both types of fibre inactivation of multi-channel Na+ currents proceeded with two time constants and late single-channel currents showed short and long openings. After application of the reducing agent glutathione to the cytoplasmic side of excised inside-out patches, inactivation of Na+ currents in both motor and sensory fibres proceeded with a single, fast exponential time constant and late currents appeared with short openings only. These data indicate that the axonal metabolism may contribute to the different inactivation kinetics of Na+ currents in motor and sensory nerve fibres

The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors

Single-channel recordings from cultured human retinal pigment epithelial cells.

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane

C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors

NMDA receptors interact via the extended intracellular C-terminal domain of the NR2 subunits with constituents of the postsynaptic density for purposes of retention, clustering, and functional regulation at central excitatory synapses. To examine the role of the C-terminal domain of NR2A in the synaptic localization and function of NR2A-containing NMDA receptors in hippocampal Schaffer collateral–CA1 pyramidal cell synapses, we analyzed mice which express NR2A only in its C-terminally truncated form. In CA1 cell somata, the levels, activation, and deactivation kinetics of extrasynaptic NMDA receptor channels were comparable in wild-type and mutant NR2A^(ΔC/ΔC) mice. At CA1 cell synapses, however, the truncated receptors were less concentrated than their full-length counterparts, as indicated by immunodetection in cultured neurons, synaptosomes, and postsynaptic densities. In the mutant, the NMDA component of evoked EPSCs was reduced in a developmentally progressing manner and was even more reduced in miniature EPSCs (mEPSCs) elicited by spontaneous glutamate release. Moreover, pharmacologically isolated NMDA currents evoked by synaptic stimulation had longer latencies and displayed slower rise and decay times, even in the presence of an NR2B-specific antagonist. These data strongly suggest that the C-terminal domain of NR2A subunits is important for the precise synaptic arrangement of NMDA receptors

Automatic Construction of Predictive Neuron Models through Large Scale Assimilation of Electrophysiological Data.

We report on the construction of neuron models by assimilating electrophysiological data with large-scale constrained nonlinear optimization. The method implements interior point line parameter search to determine parameters from the responses to intracellular current injections of zebra finch HVC neurons. We incorporated these parameters into a nine ionic channel conductance model to obtain completed models which we then use to predict the state of the neuron under arbitrary current stimulation. Each model was validated by successfully predicting the dynamics of the membrane potential induced by 20-50 different current protocols. The dispersion of parameters extracted from different assimilation windows was studied. Differences in constraints from current protocols, stochastic variability in neuron output, and noise behave as a residual temperature which broadens the global minimum of the objective function to an ellipsoid domain whose principal axes follow an exponentially decaying distribution. The maximum likelihood expectation of extracted parameters was found to provide an excellent approximation of the global minimum and yields highly consistent kinetics for both neurons studied. Large scale assimilation absorbs the intrinsic variability of electrophysiological data over wide assimilation windows. It builds models in an automatic manner treating all data as equal quantities and requiring minimal additional insight

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation

Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 μM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation. © 2018 Yang et al

A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs).

MinK and MinK-related peptide I (MiRPI) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRPI/HERG complexes reproduce the cardiac currents called I(Ks) and I(Kr), respectively. Inherited mutations in KCNEI (encoding MinK) and KCNE2(encoding MiRPI) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily