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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 586/15/3577    most recent jphysiol.2008.152314v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Wagner, L. E. Articles by Yule, D. I. PubMed PubMed Citation Articles by Wagner, L. E., II Articles by Yule, D. I. Related Collections Cellular

¹ Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, USA
² Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 230 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA

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 (P_o) 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 P_o 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.

(Received 8 February 2008; accepted after revision 3 June 2008; first published online 5 June 2008)
Corresponding author D. I. Yule: Department of Pathology, Anatomy, & Cell Biology, Thomas Jefferson University, 230 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA. Email: david_yule{at}urmc.rochester.edu