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Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy

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Abstract

Pig coronary artery smooth muscle expresses the Na+–Ca2+-exchanger NCX1 and the sarco/endoplasmic reticulum (SER) Ca2+ pump SERCA2. NCX has been proposed to play a role in refilling the SER Ca2+ pool. Caveolae may also direct Ca2+ traffic during cell signaling. Here, we use immunofluorescence microscopy to determine if there is proximity between NCX1, SERCA2, and the caveolar protein caveolin-1. Stacks of images of cell surface domains were analyzed. Image stacks for one protein were analyzed for overlap with another protein, with and without randomization or image shifting. Within the resolution of light microscopy, there is significant overlap in the distributions of NCX1, SERCA2, and caveolin-1 but the three proteins are not always co-localized. The proximity between NCX1, SERCA2 is consistent with the assertion that NCX may supply Ca2+ for refilling the SER but this relationship is only partial. Similarly, caveolae may direct traffic in some Ca2+ signaling pathways but not others.

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Abbreviations

Ca2+i:

Cytosolic Ca2+

EGTA:

Ethyleneglycol bis (b-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

NCX:

Na+–Ca2+-exchanger

PBS:

Phosphate buffered saline

PM:

Plasma membrane

PMCA:

PM Ca2+ pump

SER:

Sarco/endoplasmic reticulum

SERCA:

SER Ca2+ pump

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Acknowledgments

This work was funded by a Grant-in-aid from Heart & Stroke Foundation of Ontario (T6168, T 6355). We acknowledge Dr. E.D. Moore (University of British Columbia) for training RK in microscopy and Dr. Tony Collins (McMaster University) for guidance and suggestions.

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Authors and Affiliations

  1. Department of Medicine, McMaster University, Hamilton, ON, Canada

    Rajneet Kuner, Sue E. Samson & Ashok K. Grover

  2. Department of Biology, McMaster University, Hamilton, ON, L8N3Z5, Canada

    Iwona Kuszczak & Ashok K. Grover

Authors
  1. Iwona Kuszczak
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  2. Rajneet Kuner
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  3. Sue E. Samson
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  4. Ashok K. Grover
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Correspondence to Ashok K. Grover.

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Kuszczak, I., Kuner, R., Samson, S.E. et al. Proximity of Na+–Ca2+-exchanger and sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle: fluorescence microscopy. Mol Cell Biochem 339, 293–300 (2010). https://doi.org/10.1007/s11010-010-0392-y

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