Modulation of ionic currents in A431 human epidermoid carcinoma cells by activation of mitogen receptors

The object of my dissertation research was to evaluate the effects of mitogens on ionic currents in A431 cells. the tools used were fluorescence imaging of a membrane potential sensitive dye, di-8-ANEPPS, and whole cell patch-clamp electrophysiology. Imaging studies revealed that application of 100 $\mu$M ATP caused $\u3e$95% of the cells to hyperpolarize on the order of 30 mV. This hyperpolarization lasted for the duration of agonist presence, and was independent of extracellular calcium. Whole-cell electrophysiology was used to determine the ionic nature of the hyperpolarization. At a holding potential of $-$60 mV, ATP caused an inward current after a 4 second latency. This latency indicated a second messenger accumulation. Removal of extracellular calcium served to reduce the average magnitude of the inward current. By making various substitutions of both the intracellular and extracellular solutions, it was determined that chloride efflux made up the majority of the ATP-evoked inward current. At more depolarized holding potentials, ATP caused a transient inward current followed by a prolonged outward current. The outward current was determined to be carried by potassium, and was sensitive to charybdotoxin, which is known to block Ca$\sp{2+}$ -activated K$\sp{+}$ channels. The source of calcium was determined to be intracellular calcium pools. Inclusion of 5 mM BAPTA in the recording electrode abolished ATP activated currents. The role of G-proteins was demonstrated by the ability of GDP$\beta$S in the dialysis electrode to abolish ATP mediated currents. A dose-response study of the ATP effect revealed that at lower concentrations of ATP, fewer cells responded and that the latency prior to response was increased. The magnitude of the response, however, was independent of ATP concentration. My hypothesis for this all or none regenerative phenomenon is that a second messenger cascade causes the release of calcium internal stores. This calcium acts in a positive feedback loop to increase permeability of the plasma membrane to external calcium. The sustained rise in cytosolic calcium allows for the activation of multiple calcium dependent ion channels, including Ca$\sp{2+}$-activated K$\sp{+}$ channels, which cause an overall membrane hyperpolarization in A431 cells