Role of domain calcium in purinergic P2X2 receptor channel desensitization

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na+ with N-methyl-D-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.