Abstract
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. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether 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 the 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.
Highlights
Kv4-Dipeptidyl aminopeptidase-like protein (DPP) forms a complex of unknown stoichiometry
DPP10 Changes Kv4.2 Properties in an Expression Level-dependent Manner—DPP10 is an accessory subunit for Kv4 that is known to increase the current amplitude of Kv4.2 and to accelerate the inactivation and recovery from inactivation of Kv4.2 [38, 42, 54]
We first investigated whether the biophysical properties of Kv4.2 were variable and dependent on the expression level of DPP10 as in the case of KChIP4. 2.5 ng of Kv4.2 cRNA was injected with various amounts of DPP10 cRNA (0, 0.025, 0.25, and 2.5 ng) into Xenopus oocytes, and the currents were recorded under two-electrode voltage clamp at 1 to 3 days after the injection
Summary
Results: Kv4.2-DPP10 current properties and stoichiometry are altered depending on their relative expression levels. 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 Kv4.2DPP10 complex is variable and affects the biophysical properties of Kv4.2. 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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