Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current (I CRAC ) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits I CRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, including N,Ndimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphingosine (C 2 -ceramide) also block I CRAC . This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block I CRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit I CRAC , demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits I CRAC , and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that I CRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks I CRAC , suggesting that the sphingomyelin pathway is involved in I CRAC regulation.

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

The activation of muscarinic receptors in NIE-115 neuroblastoma cells elicits a voltage-independent calcium current. The current turns on slowly, reaches its maximum value ~ 45 s after applying the agonist, is sustained as long as agonist is present, and recovers by one half in ~ 10 s after washing the agonist away. The current density is 0.11 -+ 0.08 pA/pF (mean -+ SD; n = 12). It is absent in zero-Ca ++ saline and reduced by Mn ++ and Ba ++. The I(V) curve characterizing the current has an extrapolated reversal potential > +40 mV. The calcium current is observed in cells heavily loaded with BAPTA indicating that the calcium entry pathway is not directly gated by calcium. In fura-2 experiments, we find that muscarinic activation causes an elevation of intracellular Ca ++ that is due to both intracellular calcium release and calcium influx. The component of the signal that requires external Ca ++ has the same time course as the receptor operated calcium current. Calcium influx measured in this way elevates (Ca++)i by 89 -41 nM (n --7). Thapsigargin, an inhibitor of Ca++/ATPase associated with the endoplasmic reticulum (ER), activates a calcium current with similar properties. The current density is 0.22 ---0.20 pA/pF (n --6). Thapsigargin activated current is reduced by Mn ++ and Ba ++ and increased by elevated external Ca ++. Calcium influx activated by thapsigargin elevates (Ca++)i by 82 -+ 35 nM. The Ca ++ currents due to agonist and due to thapsigargin do not sum, indicating that these procedures activate the same process. Carbachol and thapsigargin both cause calcium release from internal stores and the calcium current bears strong similarity to calcium-release-activated calcium currents in nonexcitable cells (Hoth, M., and R.

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. (Journal of Biomolecular Screening 2005:168-181)