Classification of drugs based on properties of sodium channel inhibition: a comparative automated patch-clamp study.

BACKGROUND: There is only one established drug binding site on sodium channels. However, drug binding of sodium channels shows extreme promiscuity: ∼25% of investigated drugs have been found to potently inhibit sodium channels. The structural diversity of these molecules suggests that they may not share the binding site, and/or the mode of action. Our goal was to attempt classification of sodium channel inhibitors by measuring multiple properties of inhibition in electrophysiology experiments. We also aimed to investigate if different properties of inhibition correlate with specific chemical properties of the compounds. METHODOLOGY/PRINCIPAL FINDINGS: A comparative electrophysiological study of 35 compounds, including classic sodium channel inhibitors (anticonvulsants, antiarrhythmics and local anesthetics), as well as antidepressants, antipsychotics and neuroprotective agents, was carried out using rNav1.2 expressing HEK-293 cells and the QPatch automatic patch-clamp instrument. In the multi-dimensional space defined by the eight properties of inhibition (resting and inactivated affinity, potency, reversibility, time constants of onset and offset, use-dependence and state-dependence), at least three distinct types of inhibition could be identified; these probably reflect distinct modes of action. The compounds were clustered similarly in the multi-dimensional space defined by relevant chemical properties, including measures of lipophilicity, aromaticity, molecular size, polarity and electric charge. Drugs of the same therapeutic indication typically belonged to the same type. We identified chemical properties, which were important in determining specific properties of inhibition. State-dependence correlated with lipophilicity, the ratio of the neutral form of molecules, and aromaticity: We noticed that the highly state dependent inhibitors had at least two aromatic rings, logP>4.0, and pKa<8.0. CONCLUSIONS/SIGNIFICANCE: The correlations of inhibition properties both with chemical properties and therapeutic profiles would not have been evident through the sole determination of IC(50); therefore, recording multiple properties of inhibition may allow improved prediction of therapeutic usefulness.

Rapid desensitization of the rat α7 nAChR is facilitated by the presence of a proline residue in the outer ß-sheet.

The rat α7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the ß9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (α7-P180T) or serine (α7-P180S) slowed the onset of desensitization dramatically, with half-times of ~930 and 700 ms, respectively, compared to 90 ms for the wild-type receptor. To investigate the importance of the hydroxyl group on the position 180 side-chains, the mutant receptors α7-P180Y and α7-P180F were studied and showed half-times of desensitization of 650 and 160 ms, respectively. While a position 180 side-chain OH group may contribute to the slow desensitization rates, α7-P180S and α7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the α7-P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild-type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer ß-sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the ß10 strand. Moreover, the α7-P180T and α7-P180S mutations also formed some intrastrand H bonds along the ß9 strand, although H bonding of the OH groups of the threonine or serine side-chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild-type rat α7 nAChR is facilitated by the presence of the proline residue within the ß9 strand.

Fast- or slow-inactivated state preference of Na+ channel inhibitors: a simulation and experimental study.

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the "steady-state slow inactivation curve"), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.

Direct inhibitory effect of fluoxetine on N-methyl-D-aspartate receptors in the central nervous system.

BACKGROUND: Data accumulated in the last decade indicate that N-methyl-D-aspartate (NMDA) receptors might be involved in the pathophysiology of depression and the mechanism of action of antidepressants, although a direct inhibitory effect has been reported only in connection with tricyclic compounds, which interact with a wide range of receptors. METHODS: Using whole-cell patch-clamp recording in rat cortical cell cultures, we investigated whether the selective serotonin reuptake inhibitor fluoxetine, which has a much better adverse effect profile, has a direct effect on NMDA receptors, and we compared its action to that of the tricyclic desipramine. RESULTS: Both desipramine (concentration that causes 50% inhibition (IC(50)) = 3.13 microM) and fluoxetine (IC(50) = 10.51 microM) inhibited NMDA-evoked currents with similar efficacy in the clinically relevant low micromolar concentration range. However, in contrast to desipramine, the inhibition by fluoxetine was not voltage-dependent, and fluoxetine partially preserved its ability to associate with NMDA receptor in the presence of Mg(2+), suggesting different binding sites for the two drugs. CONCLUSIONS: The fact that different classes of antidepressants were found to be low-affinity NMDA antagonists suggests that direct inhibition of NMDA receptors may contribute to the clinical effects of antidepressants.

Analysis of finger-tapping movement.

The piano-playing-like finger-tapping movement has been analyzed with a precision image-based motion analyzer (PRIMAS). 32 healthy subjects (148 recordings) and 10 Parkinsonian patients (25 recordings) were tested. The tracking of fingers during the whole movement increased the level of information obtained from the finger-tapping test compared to visual observation or to measurement with simple contact sensors. Different feature extraction methods have been developed to evaluate the movement and thus the actual performance of the tested person. The reliability of a novel parameter, the finger-tapping test score (FTTS), that takes into account both the speed and the regularity (periodicity) of finger-tapping, was assessed in six control subjects, with four subjects tested at least 14 times. FTTS helps in staging of Parkinsonian patients. A simple and cheap device (passive marker-based analyser of movement, PAM) has been developed that is affordable for routine clinical use.

Inhibitory effect of the DA uptake blocker GBR 12909 on sodium channels of hippocampal neurons.

The effect of the selective dopamine uptake inhibitor GBR 12909 on TTX-sensitive sodium channels of cultured hippocampal neurons was investigated using whole cell patch-clamp technique. GBR 12909 dose-dependently inhibited sodium currents evoked by trains of depolarizing pulses with an IC50 of 6.3 microM. A weaker inhibition (IC50 = 17-35 microM) could be observed when currents were evoked by either single pulse depolarization or from hyperpolarized holding membrane potential. These data indicate that the extent of inhibition caused by GBR 12909 depends on the physiological activity pattern of neurons. Our results suggest that caution is needed for the interpretation of data when GBR 12909 is used for the inhibition of dopamine uptake at concentrations above the submicromolar range.