Abstract

Potassium channels are essential for many physiological functions including shaping the action potential of excitable cells and epithelial homeostasis. Their importance is highlighted by the number of diseases that result from mutations within these channels. Though they can exist as just the pore-forming alpha subunit tetramers, most, if not all, potassium channels co-assemble with non pore-forming ancillary beta subunits. These ancillary subunits function to diversify the 40 mammalian genes that account for voltage-gated potassium channel α subunits, resulting in a plethora of different potassium current signatures depending on the particular alpha and beta subunits that make up the channel. The changes imparted by the beta subunit are both alpha and beta subunit-specific. Understanding the mechanisms by which these beta subunits regulate various aspects of alpha subunit function will be important in building a model for how these channels are physiologically relevant throughout the body.

Here, we used site-directed mutagenesis followed by two-electrode voltage clamp techniques to probe the interaction between the KCNQ1 alpha subunit and extracellular acidic residues within the KCNE3 beta subunit to help understand the basis for conversion of KCNQ1 from a voltage-dependent to a largely voltage-independent channel by KCNE3. We report that aspartic acid residues presumably near the outer leaflet of the cell membrane are necessary for constitutive current at hyperpolarized potentials. We also find that these same acidic residues are responsible for preserving KCNQ1-KCNE3 current when the plasma membrane is treated with sphingomyelinase, such as occurs during Helicobacter pylori colonization of the gastrointestinal tract.

Additionally, to understand the internalization of KCNQ1 channels co-assembled with the beta subunit KCNE1, we utilized antibody-based techniques (e.g. immunoblotting and immunoprecipitation) and whole-cell patch-clamp in transiently-transfected cells. We report that the KCNE1 subunit is necessary for KCNQ1 internalization and that this process is dynamin-dependent.

The findings define important new roles for KCNE subunits and the mechanisms by which they control Kv channels such as KCNQ1. Further, the data provide insights into potential molecular etiologies of human diseases of the heart and GI tract.