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J. Biol. Chem., Vol. 279, Issue 27, 27837-27840, July 2, 2004

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Functional Mapping of Ca²⁺ Signaling Complexes in Plasma Membrane Microdomains of Polarized Cells^*

Qin Li, Xiang Luo, and Shmuel Muallem

From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040

Many cells cluster signaling complexes in plasma membrane microdomains. Polarized secretory cells cluster all Ca²⁺ signaling proteins, including GPCRs, at the apical pole. The functional significance of such an arrangement is not known because of a lack of techniques for functional mapping of signaling complexes at plasma membrane patches. In the present work, we developed such a technique based on the use of two patch pipettes, a recording and a stimulating pipette (SP). Including 20% glycerol in the SP solution increased the viscosity and the hydrophobicity to prevent leakage and formation of tight seals on the plasma membrane. This allowed moving the SP between sites to stimulate multiple patches of the same cell and with the same agonist concentrations. Functional mapping of Ca²⁺ signaling in pancreatic acinar cells revealed that the M3, cholecystokinin, and bombesin signaling complexes at the apical pole are much more sensitive to stimulation than those at the basal pole. Furthermore, at physiological agonist concentrations, Ca²⁺ signals could be evoked only by stimulation of membrane patches at the apical pole. [Ca²⁺]_i imaging revealed that Ca²⁺ waves were invariably initiated at the site of apical membrane patch stimulation, suggesting that long range diffusion of second messengers is not obligatory to initiate and propagate apical-to-basal Ca²⁺ waves. The present studies reveal a remarkable heterogeneity in responsiveness of Ca²⁺ signaling complexes at membrane microdomains, with the most responsive complexes confined to the apical pole, probably to restrict the Ca²⁺ signals to the site of exocytosis and allow the polarized functions of secretory cells.

Received for publication, April 23, 2004

^* This work was supported by National Institutes of Health Grants DE12902 and DK38938. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-648-2593; Fax: 214-648-8879; E-mail: SHMUEL.MUALLEM{at}utsouthwestern.edu.

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