Differential regulation of the InsP 3 receptor type‐1 and ‐2 single channel properties by InsP 3 , Ca 2+ and ATP

Key points•  Three family members of inositol 1,4,5‐trisphosphate receptors (InsP 3 Rs) represent ubiquitously expressed intracellular Ca 2+ release channels. The activity of the channels is regulated in a concerted and inter‐related fashion by InsP 3 , Ca 2+ and ATP. It is not established whether each isoform is regulated by these ligands in an identical fashion. In this study we directly compare the single channel activity of mammalian InsP 3 R‐1 and InsP 3 R‐2 expressed in isolation in the presence of these ligands. •  An increase in activity for each isoform was mediated by a transition from a quiescent, ‘parked’ state to a ‘drive’ mode characterized by bursting activity. Ligands did not affect the single channel activity during these bursts but instead modulate the extent of bursting activity. •  Kinetic analysis revealed that the regulation of the transition to bursting activity by Ca 2+ and ATP occurred by different mechanisms in InsP 3 R‐1 vs . InsP 3 R‐2: although the activity of both channels was biphasically regulated by Ca 2+ and changes in [InsP 3 ] did not alter this relationship, elevated ATP increased the Ca 2+ sensitivity of InsP 3 R‐1 activity without increasing the maximal achievable open probability ( P o ) of the channel. In contrast, ATP simply increased the maximal achievable P o without altering Ca 2+ sensitivity of InsP 3 R‐2. •  The differing modes of regulation of InsP 3 R‐1 and InsP 3 R‐2 probably markedly influence the characteristics of intracellular Ca 2+ signals observed in cells in which these isoforms are expressed.Abstract  An elevation of intracellular Ca 2+ levels as a result of InsP 3 receptor (InsP 3 R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP 3 R activity is tightly controlled by the levels of the primary ligands, InsP 3 , Ca 2+ and ATP. Importantly, InsP 3 Rs are regulated by in a biphasic manner. Ca 2+ release through all InsP 3 R family members is also modulated dramatically by ATP, albeit with sub‐type‐specific properties. To ascertain if a common mechanism can account for ATP and Ca 2+ regulation of these InsP 3 R family members, we examined the effects of [ATP] on the Ca 2+ dependency of rat InsP 3 R‐1 (rInsP 3 R‐1) and mouse InsP 3 R‐2 (mInsP 3 R‐2) activity expressed in DT40‐3KO cells. We used the on‐nucleus patch clamp recording technique with various [ATP], [InsP 3 ] and [Ca 2+ ] in the patch pipette and measured single InsP 3 R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP 3 ] and [ATP], the activity of rInsP 3 R‐1 and mInsP 3 R‐2 was essentially identical in terms of single channel conductance, maximal achievable open probability ( P o ) and the [Ca 2+ ] required for activation and inhibition of activity. However, in contrast to rInsP 3 R‐1 at saturating [InsP 3 ], the activity of mInsP 3 R‐2 was unaffected by [ATP]. At lower [InsP 3 ], ATP had dramatic effects on mInsP 3 R‐2 P o , but unlike the rInsP 3 R‐1, this did not occur by altering the relative Ca 2+ dependency, but by simply increasing the maximally achievable P o at a particular [InsP 3 ] and [Ca 2+ ]. [InsP 3 ] did not alter the biphasic regulation of activity by Ca 2+ in either rInsP 3 R‐1 or mInsP 3 R‐2. Analysis of the single channel kinetics indicated that Ca 2+ and ATP modulate the P o predominately by facilitating extended bursting activity of the channel but the underlying biophysical mechanism appears to be distinct for each receptor. Subtype‐specific regulation of InsP 3 R channel activity probably contributes to the fidelity of Ca 2+ signalling in cells expressing these receptor subtypes.