Effects of (−)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels

doi:10.1016/S0006-2952(01)00678-5

Biochemical Pharmacology

Volume 62, Issue 5, 1 September 2001, Pages 527-535

https://doi.org/10.1016/S0006-2952(01)00678-5 Get rights and content

Abstract

The interaction of (−)-epigallocatechin-3-gallate (EGCG), the main component of green tea (Camellia sinensis), with rat brain Kv1.5 channels (rKv1.5) stably expressed in Chinese hamster ovary (CHO) cells was investigated using the whole-cell patch-clamp technique. EGCG inhibited rKv1.5 currents at +50 mV in a concentration-dependent manner, with an ic₅₀ of 101.2 ± 6.2 μM. Pretreatment with protein tyrosine kinase (PTK) inhibitors (10 μM genistein, 100 μM AG1296), a tyrosine phosphatase inhibitor (500 μM sodium orthovanadate), or a protein kinase C (PKC) inhibitor (10 μM chelerythrine) did not block the inhibitory effect of EGCG on rKv1.5. The inhibition of rKv1.5 by EGCG displayed voltage-independence over the full activation voltage range positive to +10 mV. EGCG had no effect on the midpoint potential or the slope factor for steady-state activation and inactivation. EGCG did not affect the ion selectivity of rKv1.5. The activation (at +50 mV) kinetics was significantly slowed by EGCG. During repolarization (at −40 mV), EGCG also slowed the deactivation of the tail currents, resulting in a crossover phenomenon. Reversal of inhibition was detected by the application of repetitive depolarizing pulses and of identical double pulses, especially during the early part of the activating pulse, in the presence of EGCG. EGCG-induced inhibition of rKv1.5 showed identical affinity between EGCG and the multiple closed states of rKv1.5. These results suggest that EGCG interacts directly with rKv1.5 channels. Furthermore, by analyzing the kinetics of the interaction between EGCG and rKv1.5, we conclude that the inhibition of rKv1.5 channels by EGCG includes at least two effects: EGCG preferentially binds to the channel in the closed state, and blocks the channel by pore occlusion while depolarization is maintained.

Introduction

Green tea (Camellia sinensis) is one of the most popular beverages in the world. Because of its many scientifically proven beneficial effects on human health, it has received much attention. The protective effects of green tea against cardiovascular disease, which are induced by lowering blood lipid levels, inhibiting the oxidation of low-density lipoproteins, and inhibiting inflammatory reactions, are well documented [1], [2]. Furthermore, green tea has been reported to possess both anticarcinogenic and antitumor activities [3], [4], [5], [6]. Green tea components also play an important role as scavengers of harmful radicals [7], [8], and as antioxidants [9], [10].

Green tea contains characteristic polyphenolic compounds, commonly known as catechins: (−)-epigallocatechin-3-gallate (EGCG), (−)-epigallocatechin, (−)-epicatechin-3-gallate, and (−)-epicatechin. These compounds account for up to 30–42% of the dry weight [11]. Because EGCG is the main constituent of tea catechins, this compound has recently been investigated experimentally. Several studies have shown the molecular mechanisms of EGCG in many biological functions. EGCG inhibits PKC and protein phosphatase 2A activation via the interaction of EGCG with the phospholipid bilayer of the membrane [12]. EGCG is a selective inhibitor of tyrosine phosphorylation of PDGF-Rβ and its downstream signaling pathway [13]. These results indicate that EGCG may induce its recognized beneficial effects via a number of different mechanisms.

To our knowledge, little is known about the interaction of EGCG with any ion channel. Recent studies report that ion channels, especially K⁺ channels, are required for cell proliferation. For example, ATP-sensitive K⁺ channels are required for the progression of MCF-7 human breast cancer cells through G1 phase [14]. Furthermore, a voltage-gated K⁺ channel activity modulates Ca²⁺ influx in colon cancer cells, and subsequently modulates the proliferation of carcinoma cells in colon cancer [15]. Therefore, it is possible that EGCG induces various effects, including anticarcinogenic and antitumor activities, via a mechanism involving direct or indirect interaction between the drug and ion channels, especially K⁺ channels. We have used the voltage-gated K⁺ channel rKv1.5, expressed in CHO cells, to study its interaction with EGCG because this expression system, using a specific cloned channel, provides an important tool for the study of the pharmacological characteristics of ion channels.

The present study was undertaken to examine the effects of EGCG on rKv1.5 channels, to explore the mechanisms of EGCG action using the whole-cell patch-clamp technique, and to find a candidate mechanism with which to explain the various beneficial effects of EGCG.

Section snippets

Cell culture

The method for establishing rKv1.5 expression in CHO cells has been previously described in detail [16]. Briefly, rKv1.5 cDNA [17] was subcloned into the expression vector pCR3.1 (Invitrogen Corporation), and transfected into CHO cells using FuGENE6 (Boehringer Mannheim). Stable transfectants were selected by subculturing in the presence of G418 (Life Technologies). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Life Technologies) supplemented with 10% fetal bovine serum, 0.1

Concentration-dependent inhibition of rKv1.5 by EGCG

In whole-cell configurations, no voltage-gated current was detected in untransfected CHO cells, as described previously [19]. Fig. 1A shows the effectiveness of different EGCG concentrations on rKv1.5 currents when rKv1.5 was expressed in CHO cells. Under control conditions, rKv1.5 currents were characterized by rapid activation and then slow inactivation while a depolarizing pulse was maintained, as described previously [16]. The onset of drug action was relatively slow. When bath perfusion

Discussion

The present results show that EGCG, the main constituent of green tea, directly inhibits rKv1.5 channels in a concentration-dependent manner and independently of signal transduction pathways. Demonstration of the direct inhibition of rKv1.5 by EGCG is the first evidence for the action of this compound on ion channels.

EGCG selectively inhibits the tyrosine phosphorylation of PDGF-Rβ [13], the PTK activities of epidermal growth factor receptor, PDGF receptor, and fibroblast growth factor receptor

Acknowledgements

We thank Dr. Leonard Kaczmarek (Yale University School of Medicine, USA) for the rKv1.5 cDNA and Won Kim for reading the manuscript. This work was supported by a Korea Research Foundation Grant (KRF-2000-015-FP0026).

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¹: Abbreviations: rKv1.5, rat brain Kv1.5; EGCG, (−)-epigallocatechin-3-gallate; CHO, Chinese hamster ovary; PKC, protein kinase C; PDGF, platelet-derived growth factor; and PTK, protein tyrosine kinase.

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Effects of (−)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels1

Abstract

Introduction

Section snippets

Cell culture

Concentration-dependent inhibition of rKv1.5 by EGCG

Discussion

Acknowledgements

Biochem Pharmacol

Cancer Lett

Mutat Res

Free Radic Biol Med

Free Radic Biol Med

Biophys Chem

Life Sci

Neuron

Biochim Biopys Acta

FEBS Lett

FEBS Lett

Biophys J

Tea flavonoids and cardiovascular diseases

Crit Rev Food Sci

Influence of drinking green tea on breast cancer malignancy among Japanese patients

Jpn J Cancer Res

Cancer chemopreventive mechanisms of tea against heterocyclic amine mutagens from cooked meat

Proc Soc Exp Biol Med

Polyphenolic antioxidant (−)-epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin

Photochem Photobiol

Antioxidant chemistry of green tea catechins. Identification of products of the reaction of (−)-epigallocatechin gallate with peroxyl radicals

Chem Res Toxicol

The chemistry of tea flavonoids

Crit Rev Food Sci Nutr

Effects of (−)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels¹