Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB220

Poster Communications

The plasma membrane of Rat sub-cutaneous white adipocytes express a long Cav1.2 channel isoform

N. E. Akaniro-Ejim1, S. L. Chan1, P. A. Smith1

1. School of Life Sciences, University of Nottingham, Nottingham, Nottinghamshire, United Kingdom.

  • Figure 1: Protein expression of Cav1.2 and Cav3.1 channels in male Wistar rat sub-cutaneous adipocytes. 20μg of protein was loaded, unless otherwise specified.(A) Representative western blot of Cav1.2 and β-actin (B) Cav3.1 and β-actin expression in cytosolic (C.F.) and membrane (memb) fractions of male Wistar rat subcutaneous (s.c) adipocytes (C) Normalized signal intensity of Cav1.2 and (D) Cav3.1 expression for data shown in (A) and (B).

White fat adipocytes possess a myriad of physiological processes controlled by intracellular calcium ions (Smith et al., 2014). Although protein expression of CaV1.3 voltage-gated calcium channels (VGCCs) has been identified in white fat adipocytes (Smith et al., 2014), the expression of other VGCCS isoforms are poorly characterised. To address this question, the cytosolic and membrane fractions of Wistar rat sub-cutaneous white adipocytes was explored by western blotting. Primary adipocytes were isolated from male Wistar rat (260-360g) sub-cutaneous white adipose tissue (SC-WAT) by methods similar to those described by Bentley et al. (2014). Cytosolic and crude plasma membrane proteins were prepared by modification of the method of McKeel and Jarett (1970). Protein concentration was determined by Lowry assay and 12 - 20 µg of protein was resolved on 4 - 20% gradient precast minigels and blotted onto a nitrocellulose membrane. Samples were probed with primary antibodies (anti Cav1.2 and anti Cav3.1 - 1:500 dilution, anti β-actin - 1:5,000) and appropriate infra-red dye labelled secondary antibodies. Blots were imaged and bands expressed as the signal intensity of protein bands normalized relative to β-actin expression for each sample. Only a Cav1.2 channel isoform of > 250 kDa was detected in both cytosolic and membrane fraction of adipocytes from rat SC-WAT (Figure 1A). The expression level of this large molecular weight Cav1.2 protein was higher in the crude plasma membrane than in the cytosolic fraction (Figures 1A & C). For Cav3.1, three distinct bands of approximately 130 kDa, 70 - 75 kDa and < 70 kDa were detected, all with a slightly greater abundance in the membrane fraction relative to the cytosol (Figures 1B & D). Our data suggests not only the presence of Cav1.2 and Cav3.1 channels in rat sub-cutaneous white adipocytes, but that these exist as a variety of isoforms. Cav1.2 is a long isoform (Shistik et al., 1999, Kramer et al., 2012), preferentially expressed in the plasma membrane fraction of these cells. Whereas at least three Cav3.1 isoforms exist, whether these are proteolytic products or represent three unique proteins is not known. The functional role of these different channel isoforms in adipocytes have yet to be identified.

Where applicable, experiments conform with Society ethical requirements