Ca²⁺-activated Cl^– channels in Ehrlich ascites tumor cells are distinct from mCLCA1, 2 and3

Abstract.

Using the whole-cell patch-clamp technique, we have studied the electrophysiological and pharmacological properties of the Ca²⁺-activated Cl^– current present in Ehrlich cells. The currents activated slowly upon depolarization, deactivated upon hyperpolarization, and showed strong outward rectification. An increase in [Ca²⁺]_i activated the current with an EC₅₀ of 165.2 nM. Extracellular application of niflumic acid (100 µM) rapidly blocked the current in a voltage-dependent manner whereas sulfhydryl-modifying agents such as dithiothreitol (DTT, 1–2 mM) and N-ethylmaleimide (NEM, 100 µM) had no effect on Ca²⁺-activated currents in Ehrlich cells. Members of the recently discovered CLCA gene family are the only molecular candidates for Ca²⁺-activated Cl^– channels cloned so far. Using RT-PCR we demonstrated that the appearance of a Ca²⁺-activated Cl^– current in Ehrlich cells is not associated with the expression of the murine members of the CLCA family (mCLCA1–mCLCA3). Correspondingly, the kinetic and pharmacological properties of the Ca²⁺-activated current in Ehrlich cells differ from those of CLCA-associated currents, which are time independent and DTT sensitive. Thus, phenotypic differences in combination with RT-PCR data point to the existence of different molecular species for Ca²⁺-activated Cl^– channels.