AimJunctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca2+ entry and intracellular Ca2+ release. The small‐conductance Ca2+‐activated K+ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.MethodsAssociation of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real‐time PCR. The expression of proteins was analysed by Western blotting. Ca2+‐activated K+ current (I K,Ca) in infected adult mouse cardiac myocytes was recorded using whole‐cell voltage‐clamp technique. The intracellular Ca2+ transient was measured using an IonOptix photometry system.ResultsWe showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N‐terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I K,Ca and reduced the amplitude of the Ca2+ transient in infected cardiomyocytes.ConclusionThe present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.

Small‐conductance Ca2+‐activated K+ channel subtype2 (SK2) are stable macromolecular complexes that regulate myocardial excitability and Ca2+ homeostasis. Junctophilin‐2 (JP2) is a membrane‐binding protein, which provides functional crosstalk by physically linking with the cell‐surface and intracellular ion channels. We previously demonstrated that the MORN domain of JP2 interacts with SK2 channels. However, the roles of the JP2 MORN domain in regulating the precise subcellular localization and molecular modulation of SK2 have not yet been incompletely understood. In the present study, in vitro and in vivo assays were used to confirm the physical interactions between the SK2 channel and JP2 in H9c2 and HEK293 cells, with a concentration on the association between the C‐terminus of SK2 channels and the MORN domain of JP2. Furthermore, the membrane expression of SK2 were found to be significantly impaired by the mutation or knockdown of JP2. Using immunofluorescence staining along with Golgi/early endosome markers, we studied the mechanisms of JP2‐regulated SK2 membrane trafficking, which indicates that the JP2 MORN domain is probably necessary for the retrograde trafficking of SK2 channels. The functional study demonstrates that whole cell SK2 current densities recorded from the HEK293 cells co‐expressing the JP2‐MORN domain with SK2 were significantly augmented, compared with cells expressing SK2 alone. Our findings suggest that the MORN domain of JP2 directly modulates SK2 channel current amplitude and trafficking, through its interaction with an overlapping region of the JP2 MORN domain on the SK2 C‐terminus.

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