Kv4.2 and Accessory Dipeptidyl Peptidase-like Protein 10 (DPP10) Subunit Preferentially Form a 4:2 (Kv4.2:DPP10) Channel Complex [Molecular Biophysics]

July 24th, 2015 by Kitazawa, M., Kubo, Y., Nakajo, K.

Kv4 is a member of the voltage gated K+ channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. While DPP has been well characterized and known to increase the current amplitude and accelerate the inactivation and the recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether or not the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2/DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of DPP10 formed dimers in the plasma membrane and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2/DPP10 complex: Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2/DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2/DPP10 complex was variable depending on the relative expression level of each subunit with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.
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The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with KChIP Subunits are Variable Depending on the Relative Expression Level [Molecular Biophysics]

May 8th, 2014 by Kitazawa, M., Kubo, Y., Nakajo, K.

Kv4 is a voltage-gated K+ channel, which underlies somatodendritic subthreshold A-type current (ISA) and cardiac transient outward K+ (Ito) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits such as K+ channel interacting protein (KChIP) or dipeptidyl peptidase-like protein (DPP). KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4/KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2:KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 changes depending on the number of bound KChIP4. Subunit counting by single molecule imaging revealed that the bound number of KChIP4 in each Kv4.2/KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4/KChIP complex is variable and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.
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