Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

Abstract

A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Figure 1

CaT-L. shows Ca²⁺-dependent inactivation and binds calmodulin. (A) Representative current recorded during a voltage step to −100 mV. At least two inactivation time constants can be distinguished in 30 mM [Ca²⁺]_o. (B) Current recorded in 30 mM [Ba²⁺]_o. Note that activation is delayed, the initial fast inactivation component is missing, and currents only show a very slow second component of inactivation. (C) ³⁵S-labeled CaT-L binds to CaM-agarose in the presence, but not absence of Ca²⁺ (Left). GST-fusion proteins of CaT-L were constructed according to the schematic drawing (Right). aa, amino acid residues. (D) Binding of [³⁵S]CaM to different CaT-L GST-fusion proteins in the presence (+) or absence of calcium.

Figure 2

Properties of CaT-L's CaM binding site. (A) Sequence of the CaM binding site and corresponding regions of CaT1 (no. AAD47636) and ECaC (no. CAB40138). Line shows C4 fusion protein, hexagonal markers indicate peptide 395, and arrow points to a PKC phosphorylation site in CaT-L. Helical wheel representation of the CaM binding site (Right). (B) Dansyl-CaM fluorescence spectra in the absence or presence of 200 nM CaT-L peptide 395 at 1 mM Ca²⁺ or 2 mM EGTA. (C) Fluorescence as a function of free peptide concentration. Data points show the average of two to four experiments and the apparent K_d was 65 nM. (D) Overlay blot of GST-CaT-L C1 protein incubated with [³⁵S]CaM at different [Ca²⁺] (Upper) with the mean amount of bound CaM measured by densiometric analyses as a function of free [Ca²⁺] (n = 4; Lower).

Figure 3

Influence of phosphorylation on CaM binding to CaT-L. (A) Alignment of CaT-L's CaM binding site with PKC-α's optimal binding site (opt.) and pseudosubstrate domain (psd). (B) Fluorescence spectra of dansyl-CaM in the absence and presence of 0, 0.2, 0.4, and 1 μM phosphorylated CaT-L peptide 396. (C) Relative increase in phosphorylation of peptide 395 as a function of peptide and increasing CaM concentration using 2 nM PKC-α. (D) Autoradiographs showing phosphorylation of CaT-L protein tagged with the FLAG-epitope (Left) and of GST-C3 protein by PKC-α (Right). (E) CaM-agarose pull-down assay using wild-type and mutated CaT-L.

Figure 4

Physiological consequence of Ca²⁺-CaM binding to CaT-L. Normalized (I/I_max) current traces in response to a voltage step to −100 mV for wild-type (A), CaM binding site deletion construct S (B), point mutant construct 6 R/E (C), and disabled phosphorylation site mutant T/A (D). Note the different inactivation profiles. (E) Quantification of the residual current fraction within the first 20 ms (r20) and within the next 930 ms (r930). In the case of barium as charge carrier, residual current fraction (r930) was calculated from peak to t = 930. (F) Model of CaT-L regulation by PKC and Ca²⁺-CaM.