Computer model of unstirred layer and intracellular pH changes. Determinants of unstirred layer pH.

Transmembrane acid-base fluxes affect the intracellular pH and unstirred layer pH around a superfused biological preparation. In this paper the factors influencing the unstirred layer pH and its gradient are studied. An analytical expression of the unstirred layer pH gradient in steady state is derived as a function of simultaneous transmembrane fluxes of (weak) acids and bases with the dehydration reaction of carbonic acid in equilibrium. Also a multicompartment computer model is described consisting of the extracellular bulk compartment, different unstirred layer compartments and the intracellular compartment. With this model also transient changes and the influence of carbonic anhydrase (CA) can be studied. The analytical expression and simulations with the multicompartment model demonstrate that in steady state the unstirred layer pH and its gradient are influenced by the size and type of transmembrane flux of acids and bases, their dissociation constant and diffusion coefficient, the concentration, diffusion coefficient and type of mobile buffers and the activity and location of CA. Similar principles contribute to the amplitude of the unstirred layer pH transients. According to these models an immobile buffer does not influence the steady-state pH, but reduces the amplitude of pH transients especially when these are fast. The unstirred layer pH provides useful information about transmembrane acid-base fluxes. This paper gives more insight how the unstirred layer pH and its transients can be interpreted. Methodological issues are discussed.

Tear gasses CN, CR, and CS are potent activators of the human TRPA1 receptor.

The TRPA1 channel is activated by a number of pungent chemicals, such as allylisothiocyanate, present in mustard oil and thiosulfinates present in garlic. Most of the known activating compounds contain reactive, electrophilic chemical groups, reacting with cysteine residues in the active site of the TRPA1 channel. This covalent modification results in activation of the channel and has been shown to be reversible for several ligands. Commonly used tear gasses CN, CR and CS are also pungent chemicals, and in this study we show that they are extremely potent and selective activators of the human TRPA1 receptor. To our knowledge, these are the most potent TRPA1 agonists known to date. The identification of the molecular target for these tear gasses may open up possibilities to alleviate the effects of tear gasses via treatment with TRPA1 antagonists. In addition these results may contribute to the basic knowledge of the TRPA1 channel that is gaining importance as a pharmacological target.

P2X currents in peritoneal macrophages of wild type and P2X4 -/- mice.

In this study the ATP-induced (P2X) currents in isolated peritoneal macrophages of wild type (WT) and P2X(4) knockout (P2X(4)(-/-)) mice were studied by means of whole-cell patch clamp in order to (1) survey the P2X currents of native macrophages and (2) to investigate the expression of P2X(4)-like currents in the WT versus P2X(4)(-/-) mice. Three types of currents were observed in the isolated macrophages: (1) in approximately 10% of both WT and P2X(4)(-/-) macrophages a fast activating and inactivating P2X1-like current was recorded with low concentrations (0.1-1 microM) of ATP; (2) 85% of wild type and 100% of P2X(4)(-/-) macrophages exhibited a non-desensitizing P2X(7)-like current activated at high concentrations of ATP (10mM). The identity of the P2X(7) current was confirmed using the specific blocker A-740003; (3) 88.6% of the WT but none of the P2X(4)(-/-) macrophages showed a small P2X(4)-like current that desensitized slowly upon ATP application at intermediate concentrations (3-300 microM). Several observations indicated that the slowly desensitizing current in WT macrophages was P2X(4). The EC50 value of 5.3 microM ATP was as expected for P2X(4) and the current induced by 3-300 microM ATP was absent in P2X(4)(-/-) mice. Upon application of 3 microM ivermectin, a P2X(4)-selective modulator, the amplitude of this current was increased and the desensitization was inhibited in WT cells. In addition, this current was facilitated by 10 microM Zn(2+) but inhibited by Cu(2+) (in contrast to P2X(2)). We conclude that the P2X(4) and P2X(7) currents are functionally expressed in recruited peritoneal macrophages of WT mice and that the P2X(4)-like current is absent in P2X(4)(-/-) mice.

Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp.

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.

Computer programs to facilitate the estimation of time-dependent drug effects on ion channels.

The programs I-VOC and I-ROC have been designed to facilitate the analysis of drug effects on currents through ion channels in the cell membrane, measured by means of voltage-clamp. They are written in Igor Pro (WaveMetrics) and read exported files or raw data files generated by the Pulse (HEKA Electronic) or pClamp (Axon Instruments) programs. With I-VOC, the sweeps of current through voltage-operated ion channels can be quantified within six time ranges, and current run-down can be corrected for after the currents are fitted during the control period. Linear leak current can be subtracted even when the P/n method is not used. The results are plotted and tabulated. With I-ROC, an analogous program, receptor-operated currents can be quantified, the peak current or rate of desensitization can be fitted and run-down corrected for. Chart-like data can be converted to a sweep-like format. Several procedures are incorporated for rapid graphical data presentation. These programs accelerate and improve the estimation of drug effects on ion channels.