Regulation of human cardiac KCNQ1/KCNE1 channel by epidermal growth factor receptor kinase

Abstract

The aim of the present study was to investigate whether/how the recombinant human cardiac I(Ks) could be regulated by epidermal growth factor receptor kinase in HEK 293 cells stably expressing hKCNQ1/hKCNE1 genes using the approaches of perforated patch clamp technique, immunoprecipitation and Western blot analysis. It was found that the broad spectrum isoflavone tyrosine kinase inhibitor genistein and the selective epidermal growth factor receptor kinase inhibitor tyrphostin AG556 suppressed the recombinant I(Ks), and their inhibition was countered by the protein tyrosine phosphatase inhibitor orthovanadate. The Src-family kinase inhibitor PP2 reduced the current, but the effect was not antagonized by orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hKCNQ1 protein was decreased by genistein or AG556, but not by PP2. These results provide the novel information that epidermal growth factor receptor kinase, but not Src-family kinases, regulates the recombinant cardiac I(Ks) stably expressed in HEK 293 cells via phosphorylating KCNQ1 protein of the channel.

Apamin Does Not Inhibit Human Cardiac Na+ Current, L-type Ca2+ Current or Other Major K+ Currents

“…This cell line has relatively small endogenous currents compared to the currents expressed in transfected cells (Figure 1), making contamination by endogenous currents insignificant. HEK 293 cells have been widely used to express cloned cardiac ion channels, including Nav1.5 [44], [45], Cav1.2 [46]–[48], Kv7.1 [49], Kv11.1 [31], [50], Kir2.1 [51] and Kv4.3 [52] channels. The currents exhibited in the present experiments are consistent with those reports.…”

Genistein and tyrphostin AG556 decrease ultra‐rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase

“…Tyrosine phosphorylation of hK v 1.5 channels by EGFR kinase activates the channel and may thereby enhance the current. However, orthovanadate did not affect I Kur /hK v 1.5 current or the channel phosphorylation level, which suggests the basal tyrosine phosphorylation of hK v 1.5 channels is saturated, as observed in rat cardiac K v currentS , human cardiac I to , hERG , I Ks (Dong et al, 2010), K ir 2.1 (Zhang et al, 2011a) and also hEAG1 and hSKCa1 (Wu et al, 2013).…”

“…PTK-independent suppression by genistein has been previously observed in cardiac I Ks (Washizuka et al, 1998), I Kr (Missan et al, 2006), neuronal I Na (Liu et al, 2004), K ir 2.1 and K ir 2.3 channels (Zhao et al, 2008) and K v 4.3 channels expressed in CHO cells (Kim et al, 2011). However, PTK-dependent inhibition of human cardiac I Ks and hERG channels and hK ir 2.1, hK ir 2.3 and hK v 4.3 channels by genistein was revealed by the application of the protein tyrosine phosphatase inhibitor orthovanadate and the determination of tyrosine phosphorylation levels of these channel in our earlier reports Dong et al, 2010;Zhang et al, 2011aZhang et al, ,b, 2012. In this study, we found that genistein (3-100 μM) inhibited human atrial I Kur in a concentration-dependent manner.…”

“…Src family kinases) inhibitor PP2, and/or a protein tyrosine phosphatase inhibitor (e.g. orthovanadate) in different types of cells and investigated the regulation of a number of ion channels and currents by TKs, including L-type Ca 2+ current (I Ca.L ) in cardiac myocytes (Ogura et al, 1999), volume-sensitive chloride current (I Cl.vol ) in dog and human atrial cardiac myocytes (Sorota, 1995;Du et al, 2004), cardiac voltage-gated Na + current (I Na ) (Liu et al, 2007) and several types of K + channels in different types of cells Zhang et al, 2008Zhang et al, , 2011aZhang et al, ,b, 2012Dong et al, 2010;Wu et al, 2012Wu et al, , 2013. In the present study, the Src family kinase inhibitor PP2 slightly increased human atrial I Kur and hK v 1.5 current expressed in HEK 293 cells and significantly inhibited tyrosine phosphorylation of hK v 1.5 channels and the effects were countered by the protein tyrosine phosphatase inhibitor orthovanadate, suggesting that endogenous tyrosine phosphorylation of hK v 1.5 channels by Src family kinases would inhibit I Kur /hK v 1.5 current.…”

“…In addition, PTKs regulate transmembrane ion channels (Davis et al, 2001;Levitan, 1994). EGFR PTKs regulate several ion channels, including those carrying the cardiac voltage-gated sodium current (I Na ) (Liu et al, 2007), K ir 2.3 and K ir 2.1 (Zhang et al, 2011a,b), recombinant human cardiac I Ks (hKCNQ1/hKCNE1) (Dong et al, 2010) and the human EAG1 (K v 10.1) channels . Src family kinases also participate in regulation of hERG channels and cardiac transient outward potassium current (I to , carried by hK v 4.3 channels) ).…”