Manual patch clamps

The inhibitory potencies of three of these molecules agreed with published values for murine Kv 3 (Table 4.1). The one exception was ShK that appeared to be 10-fold less potent than both published values and our own in-house values obtained by manual patch-clamp methods. It has been reported that ShK has a slow on-rate (t= 20 min) for block of Kv 3 (Middleton et al. 2003). This phenomenon may contribute to the reduced potency of ShK in our automated electrophysiology assay, because our protocol included a compound incubation time of only 5 to 10 min. Longer compound incubation times may improve the potency of ShK but would be associated with greater run-down in the K+ current amplitude. We also tested the A LI-selective blocker, dendrotoxin, and not surprisingly it did not inhibit the Kv. 3 current at concentrations up to 167 nM, which is well above its IC50 value for Kv 1.1 in our hands (17 pM data not shown). 

The following protocol represents screening of the compounds using a manual patch clamp and the hERG channel stably transfected CHO cell line [34-36]. 

Note 4 Manual patch clamp assay is a gold standard for functional analysis of ion channels and their interactions with the drugs. Major limitation of this assay includes low through put, need of high amount of compound for testing, and requirement of highly skilled technician. The assay can be used in the late phase of drug discovery for lead optimization. 

Note 5 The assay overcomes the limitation of the manual patch clamp still the assay can be used to increase throughput during lead optimization and not during lead selection. 

Note 7 Fluorescence percentage represents the permeability of K+ channels or activity of Na+/K+ pump. The system has the potential to replace manual patch clamp or flow cytometric measurements of cardiotoxicity predictions. The potential needs to be further validated with new protocols involving testing of various cardiotoxic compounds and its direct comparison with these systems. 

The isolated oocytes are still surrounded by the vitelline membrane, an inner glycoprotein matrix layer (Yao et al., 2000). This layer does not interfere with whole-cell recording experiments. It can be removed manually however, for patch-clamp experiments requiring the formation of high-resistance seals. 

Redfern et al. studied the occurrence of QT prolongation and the lethal arrhythmia torsades de pointes (see Chapter 16) in marketed drugs from the perspective of the therapeutic index [50]. This group proposed a therapeutic index of 30-fold, calculated from the free Cmax at the therapeutic dose and the in vitro potency in the manual hERG patch clamp assay. This level, or even higher, seems to be generally followed by the industry. 

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