Korean J Physiol Pharmacol.  2020 May;24(3):277-286. 10.4196/kjpp.2020.24.3.277.

Ca2+/calmodulin-dependent regulation of polycystic kidney disease 2-like-1 by binding at C-terminal domain

• Baik JY
• Park EYJ
• So I

• KMID: 2502510
• DOI: http://doi.org/10.4196/kjpp.2020.24.3.277

Abstract

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin- L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM Nlobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu- 593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

Keyword

Calcium; Calmodulin; Ion channel; Polycystic kidney; Transient receptor potential channels

Figure

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  Fig. 1 The putative calmodulin-binding domain (CaMBD) in polycystic kidney disease 2-like-1. Protein sequence of the putative CaMBD (K590A-E600). OD, oligomerization domain.

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  Fig. 2 The effects of calmidazoluim (CMZ) on polycystic kidney disease 2-like-1 (PKD2L1) current under 16 nM and 100 nM intracellular free calcium concentrations. (A) A full current trace of PKD2L1 (left) activated by 1 μM of CMZ under 16 nM free Ca2+ and the current (I)–voltage (V) relationship of PKD2L1 (right) at the basal current amplitude (black) and at the application of 1 μM of CMZ (blue). (B) A full current trace of PKD2L1 (left) activated by 1 μM of CMZ under 100 nM free Ca2+ and the I–V relationship of PKD2L1 (right) at the basal current amplitude (black) and at the application of 1 μM of CMZ (blue). (C) A summarized current amplitude of PKD2L1 induced by CMZ under 16 nM and 100 nM free Ca2+ (n = 17–32). ***p < 0.001.

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  Fig. 3 The effects of calmidazolium (CMZ) on putative calmodulin-binding domain (CaMBD) (K590-E600) single mutants under 16 nM intracellular free calcium concentration. (A) A full current trace (left) and the current (I)–voltage (V) relationship (right) of polycystic kidney disease 2-like-1 (PKD2L1) (K590A) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (B) A full current trace (left) and the I–V relationship (right) of PKD2L1 (T591A) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (C) A full current trace (left) and the I–V relationship (right) of PKD2L1 (L593A) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (D) A summarized basal current amplitude of PKD2L1 and single mutants under 16 nM free Ca2+ (n = 4–32). (E) A summarized CMZ-induced current amplitude of PKD2L1 and single mutants under 16 nM free Ca2+ (n = 4–32). (F) A summarized current changes of PKD2L1 and single mutants by CMZ under 16 nM free Ca2+. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4 The effects of calmidazolium (CMZ) on polycystic kidney disease 2-like-1 (PKD2L1) ΔEF current under 16 nM and 100 nM intracellular free calcium concentrations. (A) A full current trace of PKD2L1 (ΔEF) (left) activated by 1 μM of CMZ under 16 nM free Ca2+ and the current (I)–voltage (V) relationship of PKD2L1 (ΔEF) (right) at the basal current amplitude (black) and at the application of 1 μM of CMZ (blue). (B) A full current trace of PKD2L1 (ΔEF) (left) activated by 1 μM of CMZ under 100 nM free Ca2+ and the I–V relationship of PKD2L1 ΔEF (right) at the basal current amplitude (black) and at the application of 1 μM of CMZ (blue). (C) A summarized current amplitude of PKD2L1 (ΔEF) induced by CMZ under 16 nM and 100 nM free Ca2+ (n = 4–5). *p < 0.05, **p < 0.01.

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  Fig. 5 The effects of calmidazolium (CMZ) on putative calmodulin-binding domain (CaMBD) (K590-E600)/ΔEF mutants under 16 nM intracellular free calcium concentration. (A) A full current trace (left) and the current (I)–voltage (V) relationship (right) of polycystic kidney disease 2-like-1 (PKD2L1) (K590A/ΔEF) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (B) A full current trace (left) and the I–V relationship (right) of PKD2L1 (T591A/ΔEF) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (C) A full current trace (left) and the I–V relationship (right) of PKD2L1 (L593A/ΔEF) activated by 1 μM of CMZ (blue) under 16 nM free Ca2+. (D) A summarized basal current amplitude of PKD2L1 (ΔEF) and mutants under 16 nM free Ca2+ (n = 4–6). (E) A summarized CMZ-induced current amplitude of PKD2L1 (ΔEF) and mutants under 16 nM free Ca2+ (n = 4–6). (F) A summarized current changes of PKD2L1 (ΔEF) and mutants by CMZ under 16 nM free Ca2+. *p < 0.05, **p < 0.01.

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  Fig. 6 The potentiation and inactivation of polycystic kidney disease 2-like-1 (PKD2L1) (L593A) with over-expression of calmodulin (CaM) under 16 nM and 100 nM intracellular free calcium concentrations. (A) A full current trace of PKD2L1 (L593A) (gray) and the mutant co-expressed with CaM (blue) under 16 nM free Ca2+. (B) A full current trace of PKD2L1 (L593A) (gray) and the mutant co-expressed with CaM (red) under 100 nM free Ca2+. (C) A summarized peak current amplitude of PKD2L1 (L593A) and the mutant co-expressed with CaM under 16 nM and 100 nM free Ca2+ (n = 4). (D) A summarized peak time of PKD2L1 (L593A) and the mutant co-expressed with CaM under 16 nM and 100 nM free Ca2+ (n = 4). *p < 0.05.

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  Fig. 7 Model of the mechanism by which calmodulin (CaM) regulates polycystic kidney disease 2-like-1 (PKD2L1). (A) In 16 nM free calcium (blue), EF-hand (yellow) constantly binds to channel and shows weak inhibition. CaM C-lobe (green) is bound to PKD2L1 through Leu-593, a CaM C-lobe anchor residue (upper panel). At this time, if EF-hand deletion occurs, PKD2L1 inhibition by EF-hand is weakened (lower panel). (B) When calcium increases to 100 nM, CaM N-lobe (orange) binds to PKD2L1 instead of EF-hand, resulting in strong PKD2L1 inhibition (upper panel). Even with EF-hand deletion, CaM N-lobe still binds to PKD2L1 and strongly inhibits its activity (lower panel). The thick line indicates strong action.

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