Differential regulation of the InsP3 receptor type‐1 and ‐2 single channel properties by InsP3, Ca2+ and ATP
Authors: Wagner II Larry E. , Yule David I. 
Published: May 2012

Key points

Three family members of inositol 1,4,5‐trisphosphate receptors (InsP3Rs) represent ubiquitously expressed intracellular Ca2+ release channels. The activity of the channels is regulated in a concerted and inter‐related fashion by InsP3, Ca2+ 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 InsP3R‐1 and InsP3R‐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 Ca2+ and ATP occurred by different mechanisms in InsP3R‐1 vs. InsP3R‐2: although the activity of both channels was biphasically regulated by Ca2+ and changes in [InsP3] did not alter this relationship, elevated ATP increased the Ca2+ sensitivity of InsP3R‐1 activity without increasing the maximal achievable open probability (Po) of the channel. In contrast, ATP simply increased the maximal achievable Po without altering Ca2+ sensitivity of InsP3R‐2.

The differing modes of regulation of InsP3R‐1 and InsP3R‐2 probably markedly influence the characteristics of intracellular Ca2+ signals observed in cells in which these isoforms are expressed.

Abstract  An elevation of intracellular Ca2+ levels as a result of InsP3 receptor (InsP3R) activity represents a ubiquitous signalling pathway controlling a wide variety of cellular events. InsP3R activity is tightly controlled by the levels of the primary ligands, InsP3, Ca2+ and ATP. Importantly, InsP3Rs are regulated by in a biphasic manner. Ca2+ release through all InsP3R 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 Ca2+ regulation of these InsP3R family members, we examined the effects of [ATP] on the Ca2+ dependency of rat InsP3R‐1 (rInsP3R‐1) and mouse InsP3R‐2 (mInsP3R‐2) activity expressed in DT40‐3KO cells. We used the on‐nucleus patch clamp recording technique with various [ATP], [InsP3] and [Ca2+] in the patch pipette and measured single InsP3R channel activity in stably transfected DT40 cells. Under identical conditions, at saturating [InsP3] and [ATP], the activity of rInsP3R‐1 and mInsP3R‐2 was essentially identical in terms of single channel conductance, maximal achievable open probability (Po) and the [Ca2+] required for activation and inhibition of activity. However, in contrast to rInsP3R‐1 at saturating [InsP3], the activity of mInsP3R‐2 was unaffected by [ATP]. At lower [InsP3], ATP had dramatic effects on mInsP3R‐2 Po, but unlike the rInsP3R‐1, this did not occur by altering the relative Ca2+ dependency, but by simply increasing the maximally achievable Po at a particular [InsP3] and [Ca2+]. [InsP3] did not alter the biphasic regulation of activity by Ca2+ in either rInsP3R‐1 or mInsP3R‐2. Analysis of the single channel kinetics indicated that Ca2+ and ATP modulate the Po 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 InsP3R channel activity probably contributes to the fidelity of Ca2+ signalling in cells expressing these receptor subtypes.