Regulation of single inositol 1,4,5‐trisphosphate receptor channel activity by protein kinase A phosphorylation

Phosphorylation of inositol 1,4,5‐trisphosphate receptors (InsP3R) by PKA represents an important, common route for regulation of Ca2+ release. Following phosphorylation of the S2 splice variant of InsP3R‐1 (S2– InsP‐1), Ca2+ release is markedly potentiated. In this study we utilize the plasma membrane (PM) expression of InsP3R‐1 and phosphorylation state mutant InsP3R‐1 to study how this regulation occurs at the single InsP3R‐1 channel level. DT40‐3KO cells stably expressing rat S2– InsP3R‐1 were generated and studied in the whole‐cell mode of the patch clamp technique. At hyperpolarized holding potentials, small numbers of unitary currents (average ∼1.7 per cell) were observed which were dependent on InsP3 and the presence of functional InsP3R‐1, and regulated by both cytoplasmic Ca2+ and ATP. Raising cAMP markedly enhanced the open probability (Po) of the InsP3R‐1 and induced bursting activity, characterized by extended periods of rapid channel openings and subsequent prolonged refractory periods. The activity, as measured by the Po of the channel, of a non‐phosphorylatable InsP3R‐1 construct (Ser1589Ala/Ser1755Ala InsP3R‐1) was markedly less than wild‐type (WT) InsP3R‐1 and right shifted some ∼15‐fold when the concentration dependency was compared to a phosphomimetic construct (Ser1589Glu/Ser1755Glu InsP3R‐1). No change in conductance of the channel was observed. This shift in apparent InsP3 sensitivity occurred without a change in InsP3 binding or Ca2+ dependency of activation or inactivation. Biophysical analysis indicated that channel activity can be described by three states: an open state, a long lived closed state which manifests itself as long interburst intervals, and a short‐lived closed state. Bursting activity occurs as the channel shuttles rapidly between the open and short‐lived closed state. The predominant effect of InsP3R‐1 phosphorylation is to increase the likelihood of extended bursting activity and thus markedly augment Ca2+ release. These analyses provide insight into the mechanism responsible for augmenting InsP3R‐1 channel activity following phosphorylation and moreover should be generally useful for further detailed investigation of the biophysical properties of InsP3R.