Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels

Levi A. Gheber,Daniel Gitler,Lior Shaltiel,Ofer Beharier,Anthony J. Muslin,Merav Mor,Liat Buzaglo,Amos Katz,Yoram Etzion,Joy Kahn,Shiri Levy,Arie Moran

doi:10.1074/jbc.m109.058842

Abstract

The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory beta-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the beta-subunit. An interaction between ZnT-1 and the beta-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming alpha(1)-subunit of the LTCC. Similarly, a decrease in the surface expression of the alpha(1)-subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC beta-subunit and that it involves a decrease in the trafficking of the LTCC alpha(1)-subunit to the surface membrane.

Highlights

Tion of cardiac excitability [1], excitation-contraction coupling [2,3,4,5], and synaptic vesicle release [6, 7]
The ␤-Subunit Is Essential for L-type calcium channel (LTCC) Inhibition by Zinc transporter 1 (ZnT-1)— We first examined whether the action of ZnT-1 depends on the presence of the ␤-subunit
Recent studies have shown that ZnT-1 is an endogenous negative regulator of the LTCC [30, 31], in the heart, where it appears to participate in cardiac electrical remodeling following atrial fibrillation [31], and in the brain, where it may affect synaptic release [37, 40]

Summary

EXPERIMENTAL PROCEDURES

Xenopus Oocytes—Xenopus laevis frogs were maintained and dissected as described previously [42]. Equal amounts (2.5 ng) of cRNAs of ␣1c, a2␦, and ␤2a with or without cRNA of ZnT-1 (2.5 ng) were injected into the oocytes as previously described [31]. Co-immunoprecipitation—Protein lysates of HEK293T cells or Xenopus oocytes expressing the ␤2a subunit and Myc-tagged ZnT-1 were prepared as described above. Thereafter, pre-washed Protein G-agarose (Santa Cruz Biotechnology) was added to the samples to pull down ZnT-1, and the mixtures were incubated at 4 °C for another 2–3 h, followed by four wash cycles with Choi lysis buffer. The following day, HEK293T cells were transfected with the ␣1- and ␤-subunits of the LTCC with or without ZnT-1 (as described above). The emission spectra were fit by the leastsquares method with the weighted average of predefined arbitrary functions describing the premeasured fluorescence spectra of the donor and acceptor, as well as of the autofluorescence of the cells. In all figures, * signifies p Ͻ 0.05, and ** p Ͻ 0.01

RESULTS

Biotinylation experiments indicate

Normalized IBa Vrev

DISCUSSION

We have previously shown that