The expression of endogenous voltage-gated potassium channels in HEK293 cells is affected by culture conditions

Abstract

HEK293 cells are widely used as a host for expression of heterologous proteins; yet, little care has been taken to characterize their endogenous membrane components, including ion channels. In this work, we aimed to describe the biophysical and pharmacological properties of endogenous, voltage‐dependent potassium currents (IKv). We also examined how its expression depends on culture conditions. We used the electrophysiological technique of whole‐cell patch clamp to record ion currents from HEK293 cells. We found that HEK cells express endogenous, voltage‐dependent potassium currents. We also found that diverse culture conditions, such as the passage number, the cell density, the type of serum that complements the culture media and the substratum, affect the magnitude and shape of IKv, resulting from the relative contribution of fast, slow, and noninactivating component currents. Incubation of cells in mature monolayers with trypsin–EDTA, notoriously reduces the magnitude and modifies the shape of voltage‐dependent potassium endogenous currents; nonetheless HEK cells recover IKv′s magnitude and shape within 6 h after replating, with a process that requires synthesis of new mRNA and protein subunits, as evidenced by the fact that actinomycin D and cycloheximide, inhibitors of synthesis of mRNA and protein, respectively, impair the recovery of IKv after trypsinization. In addition to be useful as a model expression system, HEK293 may be useful to understand how cells regulate the density of ion channels on the membrane.

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction

“…In addition, (3-(4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy) butoxy)phenyl)propyl)triphenyl phosphonium iodide (PAPTP) and (3-(((4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy)butoxy) phenoxy)carbonyl) amino) propyl) triphenylphosphonium iodide (PCARBTP), two recently described specific mitochondria-targeted derivatives of 5-(4-Phenoxybutoxy)psoralen (PAP-1), were used ( Figure 1A). PAP-1 is an inhibitor of the K + channel Kv1.3 that is expressed in different cells (Perez-Verdaguer et al, 2016), including HEK293 (Ponce et al, 2018). Both compounds were shown to inhibit the mitochondria-located Kv1.3 to trigger mitochondrial ROS release and changes in mitochondrial membrane potential (Leanza et al, 2017).…”

Optogenetically induced cellular habituation in non-neuronal cells

“…No significant decrement of the density current appeared during repetitive stimulation. In order to address any active cell-autonomous processes, we explored the impact of native potassium channels in the habituation process; we thus blocked them using 10 μM of TEA, as previously reported [14]. After confirming that TEA does not influence the photocurrent (Fig 5B), we analyzed the effect of the drug on the habituation profile at 1Hz, 5V.…”

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

“…1B, C). To limit the proportion of currents from endogenous voltage-dependent channels (Ponce et al, 2018;Yu & Kerchner, 1998), we set a minimum outward current density as an inclusion threshold for analysis (65 pA/pF at +85 mV). Of the cells patched, 7 of 18 mKv2.1 control cells and 14 of 28 mKv2.1 + AMIGO1 cells satisfied this inclusion threshold and displayed currents consistent with a Kv2.1 delayed rectifier conductance (IK) (Supplemental Fig.…”

“…Inconsistent action by auxiliary subunit(s) on a pore forming subunit counterpart is also a common problem, requiring controls to asses interaction (Jain et al, 2001;Laedermann et al, 2013;Meadows & Isom, 2005;Moran et al, 2000Moran et al, , 2003Ponce et al, 2018). Variability in channel-auxiliary subunit complexes can be due to inconsistent assembly.…”