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Although ICRAC and other store-operated currents are often analyzed by Ca(2+) imaging, whole-cell patch clamp, described here, is the preferred technique to analyze ICRAC whenever possible. The whole-cell patch-clamp protocol can even be used to record endogenous ICRAC in primary cells. The small endogenous current size of ICRAC requires some precautions: First, it is important to inhibit potential interferences from other channels in the cell by carefully choosing the combination of pipette and bath solutions. Second, the noise should be

Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.

Endogenous calcium release-activated channel (CRAC) currents are usually quite small and not always easy to measure using the patch-clamp technique. While we have, for instance, successfully recorded very small CRAC currents in primary human effector T cells, we have not yet managed to record CRAC in naïve primary human T cells. Many groups, including ours, therefore use Ca(2+) imaging technologies to analyze CRAC-dependent Ca(2+) influx. However, Ca(2+) signals are quite complex and depend on many different transporter activities; thus, it is not trivial to make quantitative statements about one single transporter, in this case CRAC channels. Therefore, a detailed patch-clamp analysis of ICRAC is always preferred. Since many laboratories use Ca(2+) imaging for ICRAC analysis, we detail here the minimal requirements for reliable measurements. Ca(2+) signals not only depend on the net Ca(2+) influx through CRAC channels but also depend on other Ca(2+) influx mechanisms, K(+) channels or Cl(-) channels (which determine the membrane potential), Ca(2+) export mechanisms like plasma membrane Ca(2+) ATPase (PMCA), sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) or Na(+)-Ca(2+) exchangers, and (local) Ca(2+) buffering often by mitochondria. In this protocol, we summarize a set of experiments that allow (quantitative) statements about CRAC channel activity using Ca(2+) imaging experiments, including the ability to rule out Ca(2+) signals from other sources.

Diese Arbeit prognostiziert mit Hilfe von finanziellen und makroökonomischen Variablen den Mehrertrag des Kanadischen S&P/TSX Venture Composite Index über 30-tägige Staatsanleihen. Anhand von Informationskriterien wird in jeder Zeitperiode das optimale Subset aus einem konstanten Basisset erklärender Variablen gewählt und für eine Schätzung verwendet. Diese Schätzung stellt die Entscheidungsgrundlage dar, aufgrund dessen das Portfolio entweder in Aktien oder Staatsanleihen gehalten wird. Durch Simulationen wird die daraus resultierende Kapitalentwicklung über den Zeitraum vom 1. Februar 2002 bis zum 30. Oktober 2009 berechnet. Dabei werden unterschiedlichste Transaktionskosten berücksichtigt. Die Resultate zeigen, dass eine rekursive Wechselstrategie in der zugrunde liegenden Zeitperiode auch bei Transaktionskosten von 0,25 Prozent eine „buy and hold“ Strategie übertrifft., Daily financial and macroeconomic variables are used to forecast one day ahead excess returns on the S&P/TSX Venture Composite Index using a recursive forecasting technique. Out of this base set of variables, different model selection criteria are used to chose an optimal subset that is used to run the forecasts at each point in time. On this basis, a rational agent decides to invest either in the stock index or in 30-day Canadian Treasury bills, depending on which asset is predicted to have a higher return. Simulations show the performance of an initial capital stock for the time horizon between 1 February 2002 and 30 October 2009, thereby considering different levels of transaction costs. The results indicate that even for 0.25 percent transaction costs the switching strategy outperforms the buy and hold strategy for the sample time period.