Abstract
KV1.5 channel function is modified by different regulatory subunits. KVβ1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVβ1.3-induced fast inactivation, decreases the amplitude of the current KV1.5–KVβ1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5–KVβ1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5–KVβ1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5–KVβ1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5–KVβ1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.
Highlights
In humans the ultrarapid outward potassium current (IKur) is only present in atria and it is generated after the activation of KV1.5 channels together with other regulatory subunits (KVβ1.2, KVβ1.3 and KVβ2.1)
We have previously demonstrated that KV1.5–KVβ1.3 channels interact with RACK1, PKCβI, PKCβII, PKCθ, PLC and PIP2, either directly or through scaffold proteins, generating a KV1.5 channelosome, observed both in HEK293 cells and in rat ventricular tissue [7], and that the inhibition of PKC reverted the pharmacological properties of KV1.5– KVβ1.3 channels towards KV1.5 channels [10]
We have reported that either inhibition of PKC or silencing the expression of PKC is able to abolish the KVβ1.3-induced fast inactivation as well as modify the pharmacology of the channel [7,10]
Summary
In humans the ultrarapid outward potassium current (IKur) is only present in atria and it is generated after the activation of KV1.5 channels together with other regulatory subunits (KVβ1.2, KVβ1.3 and KVβ2.1). We have previously demonstrated that KV1.5–KVβ1.3 channels interact with RACK1, PKCβI, PKCβII, PKCθ, PLC and PIP2, either directly or through scaffold proteins, generating a KV1.5 channelosome, observed both in HEK293 cells and in rat ventricular tissue [7], and that the inhibition of PKC reverted the pharmacological properties of KV1.5– KVβ1.3 channels towards KV1.5 channels [10]. Most PKC isoforms undergo specific compartmentation that afterwards is fine-tuned by interactions with specific PKC adaptor proteins (RACKs) [11]. The function of these adaptor proteins is to translocate bound proteins to distinct subcellular locations through their protein–protein interaction domains (WD40) [12], allowing them to regulate different cellular responses [13]. We observed that PKC inhibition both decreases the amount of KV1.5–KVβ1.3 channels in the membrane and increases their levels in the cytoplasm, suggesting that PKC activity is necessary for a correct recycling of these channels
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