Differential modulation of SK channel subtypes by phosphorylation

Abstract

Small-conductance Ca²⁺-activated K⁺ (SK) channels are voltage-independent and are activated by Ca²⁺ binding to the calmodulin constitutively associated with the channels. Both the pore-forming subunits and the associated calmodulin are subject to phosphorylation. Here, we investigated the modulation of different SK channel subtypes by phosphorylation, using the cultured endothelial cells as a tool. We report that casein kinase 2 (CK2) negatively modulates the apparent Ca²⁺ sensitivity of SK1 and IK channel subtypes by more than 5-fold, whereas the apparent Ca²⁺ sensitivity of the SK3 and SK2 subtypes is only reduced by ∼2-fold, when heterologously expressed on the plasma membrane of cultured endothelial cells. The SK2 channel subtype exhibits limited cell surface expression in these cells, partly as a result of the phosphorylation of its C-terminus by cyclic AMP-dependent protein kinase (PKA). SK2 channels expressed on the ER and mitochondria membranes may protect against cell death. This work reveals the subtype-specific modulation of the apparent Ca²⁺ sensitivity and subcellular localization of SK channels by phosphorylation in cultured endothelial cells.

Keywords: Calcium (Ca2+); Calmodulin(CaM); Casein kinase 2 (CK2); Small-conductance Ca2+-activated K+ channels (SK) cyclic AMP-dependent protein kinase (PKA).

Fig. 1.. The apparent Ca²⁺ sensitivity of SK1 and IK channel subtypes is more decreased than that of the SK3 and SK2 subtypes in immortalized endothelial cells.

Concentration-dependent activation by Ca²⁺ of the SK channel subtypes heterologously expressed in HEK293 cells (A) and immortalized endothelial cells (B). (C) EC₅₀ values for the activation by Ca²⁺, recorded from SK channel subtypes expressed in HEK293 cells (HEK) and immortalized endothelial cells (ET). All data are shown as mean ± S.E.M (n = 6–11, **P<0.01, ***P<0.001, Two-way ANOVA followed by Tukey’s post hoc tests).

Fig. 2.. The CK2 inhibitor TBB abolishes the differences between apparent Ca²⁺ sensitivity of SK channel subtypes in cultured endothelial cells.

(A) Concentration-dependent activation by Ca²⁺ of the SK channel subtypes heterologously expressed in immortalized endothelial cells in the presence of TBB (10 μM). (B) EC₅₀ values for the activation by Ca²⁺, recorded from SK channel subtypes expressed in the immortalized endothelial cells, in the absence (Control) and presence (TBB) of TBB. All data are shown as mean ± S.E.M (n = 6–9, **P<0.01, ***P<0.001, Two-way ANOVA followed by Tukey’s post hoc tests).

Fig. 3.. Subcellular localization of the SK channel subtypes in immortalized endothelial cells.

(A) Cell fractions were verified by immunoblots with antibodies for mitochondria marker (Cytochrome C), ER marker (GRP-78) and plasma membrane marker (Na⁺-K⁺ ATPase). In (B), (C) and (D), densitometry results for the subcellular markers are summarized from 3–4 experiments. (E) In cell fractions, the SK2 channel subtype is abundantly expressed on the ER membrane, in contrast to other channel subtypes. In (F), (G), (H) and (I), densitometry results for the SK channel subtypes are summarized from 4–6 experiments. All data are shown as mean ± S.E.M. *P<0.05, **P<0.01, ***P<0.001, Two-way ANOVA followed by Tukey’s post hoc tests.

Fig. 3.. Subcellular localization of the SK channel subtypes in immortalized endothelial cells.

(A) Cell fractions were verified by immunoblots with antibodies for mitochondria marker (Cytochrome C), ER marker (GRP-78) and plasma membrane marker (Na⁺-K⁺ ATPase). In (B), (C) and (D), densitometry results for the subcellular markers are summarized from 3–4 experiments. (E) In cell fractions, the SK2 channel subtype is abundantly expressed on the ER membrane, in contrast to other channel subtypes. In (F), (G), (H) and (I), densitometry results for the SK channel subtypes are summarized from 4–6 experiments. All data are shown as mean ± S.E.M. *P<0.05, **P<0.01, ***P<0.001, Two-way ANOVA followed by Tukey’s post hoc tests.

Fig. 4.. The SK2 channel subtype protects the immortalized endothelial cells against apoptosis induced by palmitate.

(A) Representative flow cytometric analysis of non-treated cells and cells treated with 80 μM palmitate using double staining with Annexin V-Alexa Fluor 647/PI. Cells in the lower right quarter indicate AnnexinV-positive, early apoptotic cells. Cells in the upper right quarter indicate AnnexinV-positive/PI-positive, late apoptotic cells. (B) Quantification of cell death (the total of early and late apoptotic cells) after 24 h treatment with palmitate (mean ± S.E.M, n = 5; **P<0.01, ***P<0.001 compared with control parent cells, Two-way ANOVA followed by Tukey’s post hoc tests).

Fig. 5.. Nano-LC/MS/MS analyses of SK2 protein isolated from the plasma membrane of the immortalized endothelial cells.

(A). The endothelial cells expressing SK2 channels were subjected to cell fractionation. SK2 channel proteins were purified from the plasma membrane fraction and then separated on SDS-PAGE gel. Protein bands at ~50 kDa and ~17 kDa correspond to SK2 channels (labeled in red box) and calmodulin, respectively. (B). The extracted ion-chromatograms of the peptide “⁵⁶⁷RSSSTAPPTSSESS⁵⁸⁰” of SK2 channels (doubly charged ion at m/z 370.80457). (C). The tandem mass spectrum of the peptide was acquired from the doubly charged precursor ion at m/z 370.80457. Fragment ion peaks as b- or y-type ions are also labeled in the spectrum. The peptide sequence including the phosphorylated sites at serines (red color) is indicated at the bottom.

Fig. 6.. SK channels are phosphorylated by kinases.

CK2 phosphorylates the CaM in complex with SK channels and leads to reduced apparent Ca²⁺ sensitivity. PKA phosphorylates the C-terminus of SK2 channels and reduces its cell surface expression. Counter-regulatory phosphatases are not shown for the purpose of clarity.