Single-channel Mean Open Time Research Articles

Alpha7 Nicotinic Receptors: Intrinsic Kinetics and Modulation by PNU 120596

We investigated the kinetics of choline evoked currents, and the effect of the positive modulator PNU 120596 on alpha7 nicotinic acetylcholine receptors expressed by GH4C1 cells (obtained from Siena Biotech SpA) in whole-cell and outside-out patch-clamp experiments. using a theta-tube system for rapid application of agonists, we measured the dependence of current kinetics on the solution exchange rate. By extrapolation and by kinetic modeling we estimated the intrinsic kinetics of the receptor: what would be the amplitude and kinetics of choline-evoked currents at instantaneous agonist application. In the presence of PNU 120596 the single channel mean open time is drastically prolonged, this allowed us to determine the ratio of simultaneously open channels using nonstationary fluctuation analysis. By determining the approximate number of channels in a patch, we could determine the peak open probability of 10 mM choline-evoked currents in the absence of the modulator (0.0333 ± 0.0056), as well as the open probability in the presence of 10 mM choline and 10 μM PNU 120596 (0.632 ± 0.065). We performed kinetic experiments to determine the affinity of PNU 120596 to three different conformational states of the receptor: resting state, desensitized state and a slowly developing second desensitized state. We found that PNU 120596 was ineffective at both the resting and the slow desensitized states, while it bound to the desensitised state and re-activated the receptors. We investigated the nature of cooperativity between the agonist and the modulator. We found that while the agonist increases the apparent affinity of the modulator only by inducing desensitized conformation (which is preferred by the modulator), the modulator induces a true increase of agonist affinity probably by allosterically affecting the conformation of the agonist binding site itself.

Simulated Block of Brain Na+ Channels by Ranolazine Predicts Selective Binding to Inactivated Conformations

The antianginal drug ranolazine inhibits the increased persistent Na+ current associated with several NaV1.1 congenital mutations causing epilepsy and migraine syndromes. This inhibition is consistent with fast-inactivated state binding. In contrast, recently published studies of ranolazine block of the cardiac NaV1.5 and skeletal muscle NaV1.4 have found evidence for pre-open and/or open state binding. In the present study, computer modeling to identify the channel states necessary and sufficient to reproduce the experimentally-observed block of brain Na+ channels by ranolazine. A minimal Markov model of a brain Na+ channel was developed that reproduces all voltage-dependent gating behaviors including activation (V½= −21.8mV), fast-inactivation (V½= −62.6mV), slow-inactivation (V½= −67.4mV), persistent current (0.23% at −10mV) and single channel mean open time (2.6msec at 0mV). In addition to three closed states, this model includes one state each for pre-open (PO), open (O), fast-inactivated (FI) and slow-inactivated (SI) states. The binding rates of ranolazine to HEK293 cells stably expressing the hNaV1.2 isoform were measured (KON= 1M−1msec−1 and KOFF= 5e−5msec−1) and were used to guide simulated binding to PO, O, FI or SI channel conformations. Only simulated binding to the FI state delayed the recovery from fast inactivation (second phase tau: control 656msec, 30uM ranolazine 729msec), which is a common feature of ranolazine block of all Na+ channel isoforms. Simulations of evoked neuronal action potential firing with 10uM ranolazine binding either to the FI or SI state demonstrated equal effectiveness for the reduction of excitability (#evoked APs during 4sec: control 174, FI binding 54, SI binding 53). No effect on AP# or frequency was observed with simulated binding to either PO or O states. Our data suggest that ranolazine binds to the inactivated states of brain Na+ channels.

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons

We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 μM capsaicin with IC 50 values in the range of 2–70 μM at a holding potential of −100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 μM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.

Regulation of single NMDA receptor channel activity by alpha-actinin and calmodulin in rat hippocampal granule cells.

The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (P(open)), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in P(open). The physiological implications of these findings are discussed.

Interaction between the sodium channel inactivation linker and domain III S4-S5

The III-IV linker (L(III-IV)) of the rat brain sodium channel is critical for fast inactivation, possibly forming a fast inactivation particle. Inactivation can be disrupted by mutation of a conserved alanine at position 1329 in the S4-S5 loop of domain III. Combination of a charged mutation at 1329 with a compensatory (opposite) charge mutation at position 1489 in L(III-IV) partially restores inactivation of the channel. The compensatory charge mutant channel has a single-channel mean open time that is similar to that of the wild-type channel and is approximately 50 times shorter than that of the L(III-IV) mutant channel. The results of thermodynamic cycle analysis indicate that the mutations in domain III S4-S5 and L(III-IV) have a coupling energy of 2.8 kcal/mol, indicating that the two mutations act interdependently. These data suggest that L(III-IV) interacts directly with A1329, which may form part of the docking site if L(III-IV) is a fast inactivation particle.

Membrane currents controlled by physical forces in cultured mesangial cells

Mechanically-activated ion channels (MACs) of cultured rat mesangial cells were stimulated by applying suction to patch pipets or by exposing cells to hypoosmotic media. MAC density was estimated as 1.5 +/- 0.4 per mu 2. In the absence of any stimulus, MAC open probabilities (N * P) were < 0.0001 increasing as a function of stretch or extracellular hypoosmolarity. Single channel mean open time during stretch increased with patch depolarization whereas hyperpolarization of the membrane delayed MAC inactivation. Ionic conductance of MACs, based on average slope conductances at hyperpolarized potentials, was 76 pS in high external K+ (N = 5) and 40 pS in high external Na+ (N = 8). PK+/PNa+ was estimated to be 4.7. MACs did not permeate Cl-, at least outwardly. Whole cell currents in response to voltage steps applied to resting cells in control conditions were approximately ohmic between -120 mV and 40 mV and were linearly and reversibly dependent on extracellular osmolarity. Our results demonstrate that: (1) MACs can be activated by both negative hydrostatic pressures applied to the pipet and by osmotic gradients; (2) MAC kinetic behavior is sensitive to membrane potential; (3) MACs may participate in cellular responses to physical forces.

Differential Development of Two Classes of Acetylcholine Receptors in Xenopus Muscle in Culture

Physiological properties of acetylcholine receptors on muscle cells at very early stages of ontogeny were compared with those of cells at later stages. Two changes were observed that contributed to an overall shortening of the mean open time of single-channels. First, there was a shift in the relative proportions of two receptor types with different conductances and mean open times, such that the contribution of receptors with large conductance and short open time increased as development proceeded. Second, there was a sharp reduction in the mean open time of channels having small conductance, with no similar change in channels having large conductance.

An apparatus for precise control of pipetting blood samples

Membrane currents controlled by physical forces in cultured mesangial cells. Mechanically-activated ion channels (MACs) of cultured rat mesangial cells were stimulated by applying suction to patch pipets or by exposing cells to hypoosmotic media. MAC density was estimated as 1.5 ± 0.4 per µ2. In the absence of any stimulus, MAC open probabilities (N * P) were < 0.0001 increasing as a function of stretch or extracellular hypoosmolarity. Single channel mean open time during stretch increased with patch depolarization whereas hyperpolarization of the membrane delayed MAC inactivation. Ionic conductance of MACs, based on average slope conductances at hyperpolarized potentials, was 76 pS in high external K+ (N = 5) and 40 pS in high external Na+ (N = 8). PK+/PNa+ was estimated to be 4.7. MACs did not permeate Cl-, at least outwardly. Whole cell currents in response to voltage steps applied to resting cells in control conditions were approximately ohmic between -120 mV and 40 mV and were linearly and reversibly dependent on extracellular osmolarity. Our results demonstrate that: (1) MACs can be activated by both negative hydrostatic pressures applied to the pipet and by osmotic gradients; (2) MAC kinetic behavior is sensitive to membrane potential; (3) MACs may participate in cellular responses to physical forces.