Objectives: 

Calcium is a second messenger involved in many cellular processes including cell proliferation. Critical keys in this regard are the store-operated calcium channels activated by calcium release from intracellular stores and modulated by mitochondria. The molecular identity, biophysical properties and physiological functions of store-operated currents (SOCs) in different cell types are still not clear. Patch-clamp electrophysiology is an excellent technique for studying ion channels, but the extremely low single conductance (~fS) of SOCs hampers their characterization. The present work has focused on electrical recording of SOCs among different cells using an automated patch-clamp device.

Materials: 

A port-a-patch planar patch-clamp system (Nanion Technologies) was used for high resolution electrophysiological recordings in whole-cell configuration. Using borosilicate-glass chips, we were able to achieve seals exceeded 1 giga-ohm and recorded suspended cells including pituitary, vascular smooth muscle and colonic cells. SOCs were measured by applying voltage ramps (-100 to +100 mV) from a holding potential of 0 mV. For activation, intracellular calcium stores were depleted by chelating cytosolic calcium, intracellular thapsigargin and IP3 and external thapsigargin.

Results: 

SOCs were found in all cell lines studied (around 1-10 pA/pF). I/V relationships were characterized by small inward current amplitude, voltage-independent activation, inward rectification, and reversal potential in positive voltages. High intracellular EGTA decreased slow inactivation and amplitude was reduced by 2APB.

Conclusions: 

In all cell lines examined SOCs could be accurately examined with planar patch-clamp. The recordings obtained allow us preliminary biophysical and pharmacological characterization of SOCs that may play important roles in these cells, particularly in cell proliferation.

This work was funded by DIGICYT (BFU2009-08967) and Junta de Castilla y León, Spain (VA270A11-2).