Characterization of whole-cell currents in mucosal and connective tissue rat mast cells using amphotericin-B-perforated patches and temperature control

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Abstract
Rat mucosal type mast cells are thought to possess only a K+-selective inwardly rectifying (IRK) current in the resting state. We used rat-bone-marrow-derived mast cells (BMMCs) as a model of mucosal mast cells and recorded whole-cell membrane currents from cells perforated with amphotericin B. Under these conditions, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed. The reversal potential and conductance of the IR current depended on the extracellular K+ concentration, indicating that the channel was K+ selective. The OR current was not affected by changes in extracellular K+ concentration, but lowering extracellular Cl− concentration reduced the conductance and shifted the reversal potential in a positive direction. The OR current was not affected by K+ channel blockers, but was reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanato-2,2′-stilbenedisulphonate (DIDS), again indicating a Cl− conductance. The IRK current was also detected in the majority of cells using the conventional whole-cell recording configuration at room temperature. In contrast, the ORC1 current was only observed in 7% of recordings made at room temperature with the conventional whole-cell voltage-clamp mode, but was detected in 66% of cells if the bath temperature was increased and the integrity of the cell’s cytoplasm was preserved by using the perforated-patch technique. Under similar conditions, the ORC1 current was also present in rat peritoneal mast cells, a connective tissue phenotype previously thought to have no whole-cell currents in the resting state. The role of this current and factors affecting its activation are discussed.