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

Phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP 3 R) by PKA represents an important, common route for regulation of Ca 2+ release. Following phosphorylation of the S2 splice variant of InsP 3 R-1 (S2– InsP-1), Ca 2+ release is markedly potentiated. In this study we utilize the plasma membrane (PM) expression of InsP 3 R-1 and phosphorylation state mutant InsP 3 R-1 to study how this regulation occurs at the single InsP 3 R-1 channel level. DT40-3KO cells stably expressing rat S2– InsP 3 R-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 InsP 3 and the presence of functional InsP 3 R-1, and regulated by both cytoplasmic Ca 2+ and ATP. Raising cAMP markedly enhanced the open probability ( P o ) of the InsP 3 R-1 and induced bursting activity, characterized by extended periods of rapid channel openings and subsequent prolonged refractory periods. The activity, as measured by the P o of the channel, of a non-phosphorylatable InsP 3 R-1 construct (Ser1589Ala/Ser1755Ala InsP 3 R-1) was markedly less than wild-type (WT) InsP 3 R-1 and right shifted some ∼15-fold when the concentration dependency was compared to a phosphomimetic construct (Ser1589Glu/Ser1755Glu InsP 3 R-1). No change in conductance of the channel was observed. This shift in apparent InsP 3 sensitivity occurred without a change in InsP 3 binding or Ca 2+ 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 InsP 3 R-1 phosphorylation is to increase the likelihood of extended bursting activity and thus markedly augment Ca 2+ release. These analyses provide insight into the mechanism responsible for augmenting InsP 3 R-1 channel activity following phosphorylation and moreover should be generally useful for further detailed investigation of the biophysical properties of InsP 3 R.