Estimating neuronal conductance model parameters using dynamic action potential clamp.

BACKGROUND: Parameterization of neuronal membrane conductance models relies on data acquired from current clamp (CC) or voltage clamp (VC) recordings. Although the CC approach provides key information on a neuron's firing properties, it is often difficult to disentangle the influence of multiple conductances that contribute to the excitation properties of a real neuron. Isolation of a single conductance using pharmacological agents or heterologous expression simplifies analysis but requires extensive VC evaluation to explore the complete state behavior of the channel of interest. NEW METHOD: We present an improved parameterization approach that uses data derived from dynamic action potential clamp (DAPC) recordings to extract conductance equation parameters. We demonstrate the utility of the approach by applying it to the standard Hodgkin-Huxley conductance model although other conductance models could be easily incorporated as well. RESULTS: Using a fully simulated setup we show that, with as few as five action potentials previously recorded in DAPC mode, sodium conductance equation parameters can be determined with average parameter errors of less than 4% while action potential firing accuracy approaches 100%. In real DAPC experiments, we show that by "training" our model with five or fewer action potentials, subsequent firing lasting for several seconds could be predicted with ˜96% mean firing rate accuracy and 94% temporal overlap accuracy. COMPARISON WITH EXISTING METHODS: Our DAPC-based approach surpasses the accuracy of VC-based approaches for extracting conductance equation parameters with a significantly reduced temporal overhead. CONCLUSION: DAPC-based approach will facilitate the rapid and systematic characterization of neuronal channelopathies.

Discovering the pharmacodynamics of conolidine and cannabidiol using a cultured neuronal network based workflow.

Determining the mechanism of action (MOA) of novel or naturally occurring compounds mostly relies on assays tailored for individual target proteins. Here we explore an alternative approach based on pattern matching response profiles obtained using cultured neuronal networks. Conolidine and cannabidiol are plant-derivatives with known antinociceptive activity but unknown MOA. Application of conolidine/cannabidiol to cultured neuronal networks altered network firing in a highly reproducible manner and created similar impact on network properties suggesting engagement with a common biological target. We used principal component analysis (PCA) and multi-dimensional scaling (MDS) to compare network activity profiles of conolidine/cannabidiol to a series of well-studied compounds with known MOA. Network activity profiles evoked by conolidine and cannabidiol closely matched that of ω-conotoxin CVIE, a potent and selective Cav2.2 calcium channel blocker with proposed antinociceptive action suggesting that they too would block this channel. To verify this, Cav2.2 channels were heterologously expressed, recorded with whole-cell patch clamp and conolidine/cannabidiol was applied. Remarkably, conolidine and cannabidiol both inhibited Cav2.2, providing a glimpse into the MOA that could underlie their antinociceptive action. These data highlight the utility of cultured neuronal network-based workflows to efficiently identify MOA of drugs in a highly scalable assay.

Effects of arginine 10 to lysine substitution on ω-conotoxin CVIE and CVIF block of Cav2.2 channels.

BACKGROUND AND PURPOSE: ω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs). EXPERIMENTAL APPROACH: Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively. KEY RESULTS: R10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ω-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID. CONCLUSIONS AND IMPLICATIONS: R10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Membrane orientation of droplets prepared from Chara corallina internodal cells.

It is generally accepted that the membrane surrounding droplets from characean cells originates from the tonoplast, but there is some uncertainty regarding droplet membrane sidedness. This issue was addressed directly by combining two different droplet isolation methods and the patch clamp technique. Neutral red accumulation was used to demonstrate the presence of H(+)-transport over the membrane and to predict membrane orientation. Two types of droplet populations with differently oriented membranes could be formed in an iso-osmotic bath solution. Cytoplasmic droplets (cytosolic side of the tonoplast inside) contained cytoplasm, while the second type of droplet population contained vacuolar sap (vacuolar droplets, vacuolar side of the tonoplast inside). Smaller vesicels also appeared inside the droplets, with an apparently inversely oriented membrane. Confocal laser scanning microscopy indirectly demonstrated that, at least with one of the droplet isolation methods, the plasma membrane entirely remains in the internodal cell after intracellular perfusion. Both types of droplet populations allowed the formation of excised patches and single-channel measurements by the patch clamp technique. Properties of anion channels in the tonoplast could be used to prove the predicted membrane orientation, knowing that Ca2+ can only activate these channels from the cytosolic side. These results provide useful data for studies addressing ligand-binding, block and modulation, organization and interaction of proteins within the membrane or with other regulatory factors, where it is important to control membrane orientation.

Tonoplast anion channel activity modulation by pH in Chara corallina.

The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca2+-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pH(vac) 5.3 or 6.0) and a cytosolic pH (pH(cyt)) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In addition, Ca2+-sensitivity of the channels was markedly influenced by pH changes. At pH(cyt) values of 7.2 and 7.4 the half-maximal concentration (EC50) for calcium activation was 100-200 microm, whereas an EC(50) of about 5 microm was found at a pH(cyt) of 6.0. This suggests an improved binding of Ca2+ ions to the channel protein at more acidic cytoplasm. At low pH(cyt), anion channel activity and mean open times were voltage-dependent. At pipette potentials (V(p)) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean open time of the channel increased. In contrast, at pH(cyt) 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V(p). The kinetics of the channel could be further controlled by the Ca2+ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic Ca2+ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range, below the resting pH(cyt). A putative physiological role of the pH-dependent modulation of these anion channels is discussed.

The presence and subcellular localization of caspase 3-like proteinases in plant cells.

Caspases play a very important role in initiating and executing apoptotic processes in animal cells. In this study we show that plant mitochondria were able to initiate the activation of caspase 3 in a Xenopus cell free system. Caspase 3-like activity was found to be present in plant cells and could only be inhibited by the specific caspase 3 inhibitor N-acetyl-Asp-Glu-Val-Asp-fluoromethylketone (Ac-DEVD-fmk) and not by cysteine protease inhibitors. By micro-injection of the caspase 3 substrate in living Chara cells we showed that caspase 3-like activity was mainly present in the cytosol rather than in the vacuole. This is the first time that in vivo caspase 3-like activity has been demonstrated in plants.

Anion channels in chara corallina tonoplast membrane: calcium dependence and rectification.

Tonoplast K(+) channels of Chara corallina are well characterized but only a few reports mention anion channels, which are likely to play an important role in the tonoplast action potential and osmoregulation of this plant. For experiments internodal cells were isolated. Cytoplasmic droplets were formed in an iso-osmotic bath solution according to a modified procedure. Ion channels with conductances of 48 pS and 170 pS were detected by the patch-clamp technique. In the absence of K(+) in the bath solution the 170 pS channel was not observed at negative pipette potential values. When Cl(-) on either the vacuolar side or the cytoplasmic side was partly replaced with F(-), the reversal potential of the 48 pS channel shifted conform to the Cl(-) equilibrium potential with similar behavior in droplet-attached and excised patch mode. These results showed that the 48 pS channel was a Cl(-) channel. In droplet-attached mode the channel rectified outward current flow, and the slope conductance was smaller. When Chara droplets were formed in a bath solution containing low (10(-8) m) Ca(2+), then no Cl(-) channels could be detected either in droplet-attached or in inside-out patch mode. Channel activity was restored if Ca(2+) was applied to the cytoplasmic side of inside-out patches. Rectification properties in the inside-out patch configuration could be controlled by the holding pipette potential. Holding potential values negative or positive to the calculated reversal potential for Cl(-) ions induced opposite rectification properties. Our results show Ca(2+)-activated Cl(-) channels in the tonoplast of Chara with holding potential dependent rectification.

Peripheral blood lymphocytes display reduced K+ channel activity in aged humans.

Steady-state parameters of whole-cell K+ current have been determined in human peripheral blood lymphocytes of young (20-50 y.) and elderly (> 90 y.) volunteers by patch-clamp. The magnitude and voltage dependence of the K+ conductance were similar in both lymphocyte populations. The midpoint of steady-state inactivation was -53.3 +/- 2.3 mV for lymphocyte population of young individuals and -65.0 +/- 3.0 mV for that of elderly, showing a significant shift to hyperpolarized potentials. The peak of the steady-state open probability of the K+ channels was decreased and shifted to depolarized potentials by approx. 12.5 mV for lymphocytes of elderly donors. It is suggested that the observed differences in the K+ current parameters may be at least partly responsible for the impaired responsiveness of elderly lymphocytes to proliferative stimuli.