Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.

Inhibition of cardiac potassium currents by pentobarbital.

The inhibitory effects of the anesthetic barbiturate pentobarbital on the slow ( I(Ks)) and fast component ( I(Kr)) of cardiac delayed rectifier potassium currents ( I(K)) and on the inward rectifier potassium currents ( I(K1)) were examined in Xenopus oocytes expressing the human minK, human ether-á-go-go related gene (HERG) and guinea pig Kir2.2, respectively. Block of native I(K) ( I(Ks) and I(Kr)) and inward rectifier potassium current ( I(K1)) by pentobarbital was examined in guinea pig ventricular myocytes. In oocytes using the two electrode voltage clamp technique potassium currents of hminK-, HERG- and Kir2.2-expressing oocytes were inhibited by pentobarbital with IC50 values of 0.20, 1.58 and 0.54 mM, respectively. I(Ks) block was time- and voltage-independent and had no influence on activation at positive voltages although it shifted voltage-dependent activation to more positive voltages. Pentobarbital-induced HERG inhibition was not dependent on voltage but influenced the deactivation kinetics and shifted half-maximal activation to more negative voltages. In guinea pig cardiomyocytes, using the patch clamp technique, I(Ks) and I(Kr) were inhibited by pentobarbital with IC50 values of 0.18 mM and 2.75 mM, respectively. I(Kr) deactivation and I(Ks) activation kinetics were only slightly influenced by pentobarbital, if at all. Block of I(K1) was weakly voltage-dependent with IC(50) values of 0.26 mM (-40 mV) and 0.91 mM (-120 mV). The data show that pentobarbital suppresses both cloned ( I(K), I(Kir2.2)) and native ( I(K), I(K1)) cardiac potassium currents with the highest affinity for I(Ks).