Canonical Bcl-2 Motifs of the Na+/K+ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?

Abstract

Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na⁺/K⁺ pump/ATPase [NKP, NKA], Na⁺-K⁺-2Cl⁺ [NKCC] and K⁺-Cl^- [KCC] cotransporters, and channel-supported K⁺ loss) in human lens epithelial cells [LECs]. Methods: K⁺ loss and K⁺ uptake, using Rb⁺ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl^- replacement by NO₃^ˉ to determine KCC. ³H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by >90% (IC₅₀ values ∼35 and ∼15 µM, respectively) without significant KCC activity change, and stimulated K⁺ loss by ∼35% at 10-30 µM. Neither ATP levels nor phosphorylation of the NKA α1 subunit changed. ³H-ouabain was displaced from pig renal NKA only at 100 fold higher CET concentrations than the ligand. Sequence alignments of NKA with BH1- and BH3-like motifs containing pro-survival Bcl-2 and BclXl proteins showed more than one BH1-like motif within NKA for interaction with CET or with BH3 motifs. One NKA BH1-like motif (ARAAEILARDGPN) was also found in all P-type ATPases. Also, NKA possessed a second motif similar to that near the BH3 region of Bcl-2. Conclusion: Findings support the hypothesis that CET inhibits NKP by binding to BH1-like motifs and disrupting the α₁ subunit catalytic activity through conformational changes. By interacting with Bcl-2 proteins through their complementary BH1- or BH3-like-motifs, NKP proteins may be sensors of normal and pathological cell functions, becoming important yet unrecognized signal transducers in the initial phases of apoptosis. CET action on NKCC1 and K⁺ channels may involve PKC-regulated mechanisms; however, limited sequence homologies to BH1-like motifs cannot exclude direct effects.