Functional interaction of Junctophilin 2 with small- conductance Ca²⁺ -activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

Aim: Junctophilins (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.

Methods: Association 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.

Results: We 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.

Conclusion: The 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.