Stimulation of N-Terminal Truncated Isoform of Androgen Receptor Stabilizes Human Ether-á-go-go-Related Gene-Encoded Potassium Channel Protein via Activation of Extracellular Signal Regulated Kinase 1/2

Abstract

Proarrhythmic drugs induce long QT syndrome more frequently in women than men. The present study was designed to determine whether androgens regulate the function and expression of the human ether-á-go-go-related gene (HERG) encoded K+ channel, which is largely responsible for determining the QT interval. In a concentration-dependent manner (10(-9) to 10(-6) M for 24 h), 5alpha-dihydrotestosterone (5alpha-DHT) increased HERG protein abundance in HEK293 cells stably expressing HERG in the presence of coexpressed cardiac androgen receptor (AR) variant [N-terminal truncated isoform of AR (AR45)]. The elevation of HERG protein was seen in endoplasmic reticulum, Golgi, and plasma membrane without clear preferential colocalization. Coexpression of the more common form of the AR did not confer 5alpha-DHT augmentation of HERG protein. Proteasome inhibitors, N-acetyl-L-leucyl-L-leucyl-L-norleucinal and MG132 prevented the 5alpha-DHT- dependent enhancement of HERG, as did the lysosome inhibitor, bafilomycin A1. Consistently, the cycloheximide-based protein chase study showed that 5alpha-DHT prolonged HERG protein half-life. 5alpha-DHT/AR45 signaling induced phosphorylation of ERK1/2. Blockade of ERK1/2 with PD98059 and U0126 prevented the effect of androgen on HERG protein abundance. Functional studies showed that 5alpha-DHT treatment for 24 h increased HERG K+ current density in Chinese hamster ovary cells cotransfected with cDNAs of AR45 and HERG channels. Moreover, 5alpha-DHT also increased ether-á-go-go-related gene-encoded K+ channel protein abundance in isolated rabbit cardiac myocytes. In conclusion, these data provide evidence that stimulation of AR45 receptors by androgens up-regulates HERG K+ channel abundance and activity mainly through stabilizing HERG protein in an ERK1/2 dependent mechanism, and suggest a mechanism to explain the sex difference in the long QT syndrome.

Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis

“…CHO cells were transiently transfected with cDNAs of HERG and GFP by electroporation as described previously (21). To study the L-type Ca 2ϩ current, HEK293 cells were transiently transfected with human Ca v 1.2 cardiac splicing form Ca v 1.2CM (1a-8a-32-33) constructs (1.25 g), together with ␤2a (1.25 g) and ␣2␦ (1.25 g) using the calcium phosphate transfection method (22).…”

“…Cells were lysed as described previously (21 Western Blot Analysis-Western blot analysis was performed using whole cell lysates as described previously (21). Anti-myc mouse monoclonal 9E10 and anti-tubulin rabbit monoclonal antibodies were from Sigma.…”

“…Confocal Microscopy-HERG-HEK293 cells grown on glass coverslips were subject to formaldehyde fixation before indirect immunofluorescent staining. The method was described in our previous publication (21).To study the plasma membrane HERG K ϩ channel distribution, cells were incubated with anti-K v 11.1 (HERG extracellular epitope, residues 430 -445, between S1 and S2 loop) antibody (Alomone Labs, Jerusalem, Israel) without permeabilization, and the primary antibody was detected with Alexa Fluor 488-conjugated anti-rabbit Ig (Invitrogen).…”

“…Patch Clamp Recording-Cells grown on coverslips were placed in an acrylic/polystyrene perfusion chamber (Warner Instruments) for electrophysiological measurements and are described in our previous publications (21,22). For whole cell voltage clamp, the pipette solution consisted of 126 mM KCl, 2 mM MgSO 4 , 0.5 mM CaCl 2 , 5 mM EGTA, 4 mM MgATP, and 25 mM Hepes (pH 7.2, osmolality: 280 Ϯ 10 mOsm).…”

Coregulation of srGAP1 by Wnt and Androgen Receptor Signaling: A New Target for Treatment of CRPC

“…An AR variant [N-terminal truncated isoform of AR (AR45)], with an altered N-terminal domain with a replacement by a unique, short, seven amino-acid-long stretch, has been identified [57]. Overexpression of AR45 was shown to interact with the full-length AR and inhibit AR transcriptional activity and inhibit the growth of LNCaP cells [58].…”