Elsevier

Fish & Shellfish Immunology

Volume 33, Issue 3, September 2012, Pages 605-613
Fish & Shellfish Immunology

Short communication
Molecular cloning and functional analysis of a voltage-gated potassium channel in lymphocytes from sea perch, Lateolabrax japonicus

https://doi.org/10.1016/j.fsi.2012.05.024Get rights and content

Abstract

Voltage-gated potassium (Kv) channels on cell plasma membrane play an important role in both excitable cells and non-excitable cells and Kv1 subfamily is most extensively studied channel in mammalian cells. Recently, this potassium channel was reported to control processes inside mammalian T lymphocytes such as cell proliferation and volume regulation. Little is known about Kv1 channels in fish. We have postulated the presence of such a channel in lymphocytes and speculated its potential role in immunoregulation in fish. Employing specific primers and RNA template, we cloned a segment of a novel gene from sea perch blood sample and subsequently obtained a full cDNA sequence using RACE approach. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel Kv channel gene, designated as spKv1.3, which exhibits homologous domains to the members of Kv1.3 family, but it differs notably from some other members of that family at the carboxyl terminus. Full-length of spKv1.3 cDNA is 2152 bp with a 1440 bp open reading frame encoding a protein of 480 amino acids. SpKv1.3 gene is expressed in all of the tested organs and tissues of sea perch. To assess the postulated immune function of spKv1.3, we stimulated lymphocytes with LPS and/or channel blocker 4-AP. Expression levels of messenger RNA (mRNA) of spKv1.3 under stimulation conditions were measured by quantitative RT-PCR. The results showed that LPS can motivate the up-regulation of spKv1.3 expression significantly. Interestingly, we found for the first time that 4-AP with LPS can also increase the spKv1.3 mRNA expression levels in time course. Although 4-AP could block potassium channels physically, we speculated that its effect on blockage of potassium channel may start up an alternative mechanism which feed back and evoke the spKv1.3 mRNA expression.

Highlights

► For the first time, the full length of Kv1.3 gene was gotten from Japanese sea perch. ► spKv1.3 was a constitutive and inducible acute-phase gene in sea perch lymphocytes. ► LPS can increase the expression level of spKv1.3 mRNA. ► We found 4-AP+LPS can increase the spKv1.3 mRNA expression levels in time course. ► spKv1.3 genomic DNA was intronless.

Introduction

Voltage-gated potassium (Kv) channels play a crucial role in excitable cells, determining the resting membrane potential and controlling action potentials [1]. In addition, some observations indicate that they are involved in the control and modulation of cell functions in non-excitable cells [2]. Plentiful researches on the role of potassium channels in cell functional regulation in non-excitable cells came from experiments on mammalian lymphocytes. Available data provide evidence that delayed rectifier potassium channels and calcium-activated potassium channels are present in the lymphocytes plasma membrane of diversiform vertebrates [3], [4]. However, evidence demonstrated that Kv channels are predominant in resting human lymphocytes [3]. It has been shown that Kv channels play an important role in setting the lymphocytes membrane resting potential [5]. Moreover, these channels are involved in multiple cell functions inside lymphocytes such as mitogenesis [6], [7] and volume regulation [8].

The discovery of Kv channels in lymphocyte has led to a rapidly growing body of knowledge about their functional roles in the immune system. The most important one among these Kv channels is the n-type channel, which is encoded by the Kv1.3 gene, and therefore, it has been referred to as the Kv1.3 channel within the Shaker family of Kv channels. Kv1.3 performs a key function in innate immune system, initiating and controlling cytokine synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL) [9]. The Kv1.3 channel was first identified by Matteson and Deutsch [10]and DeCoursey et al [6], afterwards it was cloned from human lymphocytes by Grissmer et al. [11]. Structurally it is assembled from four identical, non-covalently linked subunits. Each subunit contains six transmembrane segments (S1–S6) connected by intra- and extracellular loops. The S4 segment is functioning as the voltage sensor, this segment contains repeated motifs of a positively charged residue followed by two hydrophobic residues. Identification of the voltage-sensors and gating charges of the voltage-gated ion channels is the first critical step toward understanding of the molecular basis of voltage-dependent activation. The extracellular loop between the fifth and sixth transmembrane segments (S5 and S6) along with segments of the S6 helix form the pore through which potassium ions cross the membrane. This region also serves as a receptor for inhibitor of the channel. The pore region is highly conserved across the majority of K+ channels since it contains the selectivity filter that confers the high selectivity for potassium ions over other ion species [8].

LPS is a principal component of the outer membrane of Gram-negative bacteria. Several types of cells, such as monocytes and lymphocytes, orchestrate the innate immune response to LPS by expressing a variety of inflammatory cytokines that include TNF-α and IL [9], [12]. Evidence has shown that the Kv channel blockers, TEA, barium chloride and quinine, can inhibit LPS-induced TNF release from mammalian macrophages [13]. The drug 4-aminopyridine (4-AP) is known to block LPS-induced Kv channels presented in variety of cell types including lymphocytes [14]. The K+ channels in T lymphocytes can be blocked by 4-AP with one-to-one drug molecule-channel stoicheiometry [15], which means the blockade of potassium channels by 4-AP is dose-dependent. Until now, little is known about the function of Kv channels in immune response and whether 4-AP can inhibit LPS-induced Kv channels in marine fish.

The Japanese sea perch is of great interest for fisheries as well as aquaculture because of its highly commercial value. It has become one of the main marine commercial fish species, and is popularly cultured in China. With rapid expansion of sea perch commercial aquaculture in recent years, disease control and immune regulation studies on this species have become critical for sustainable development of aquaculture industry. Understanding of ion channels function will provide a new angle on the mechanisms of immune response to pathogen invasion. Pharmacological tools open the way toward the understanding of the role of K+ channels in cell proliferation but are limited because of their lack of specificity when the identification of the individual K+ channels was attempted. Furthermore, pharmacological approaches often fail to give deep insight into the molecular mechanisms [16]. More direct evidence regarding the role of K+ channels on immune response will be possible after the molecular entities underlying K+ currents make clear. Here, we report, for the first time, the cloning and characterization of spKv1.3 gene isolated from sea perch, Lateolabrax japonicus, and first time study the function of Kv1.3 channel in fish. We provide information about if LPS could affect the mRNA expression of spKv1.3 gene and the behavior of this gene after channel blocker treatment.

Section snippets

Animals

Healthy sea perch (800 g mean weight) were obtained from an aquaculture farm in Qingdao (Shandong, China) and kept in a 260 L seawater tank at 25 °C for later studies. Blood, brain, head kidney, liver, spleen, intestines, gills, muscle and skin samples were isolated from the fish. Total RNA was prepared from those tissues using standard TRIzol Reagent (Invitrogen, California, USA).

Cell separation and culture

Peripheral blood was collected by puncturing caudal vein of the sea perch individual using a heparinized syringe.

Sea perch spKv1.3 cDNA sequence

The sea perch voltage-gated potassium channel cDNA segment was amplified from the lymphocytes of sea perch. The segment was 629 bp and predicted to be the core region of a voltage-gated potassium channel subfamily A, member 3 gene by Blast. Taking this segment as a template, two cDNA fragments of 765 bp and 1144 bp were obtained through 5′RACE and 3′RACE approaches respectively. The full length of cDNA contained an open reading frame (ORF) of 1440 bp with 5′ untranslated region (UTR) of 230 bp

Acknowledgement

This work was financially supported by Chinese National Natural Science Foundation Grant No: 40876089 and 41006102.

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      Homeostatic plasticity of intrinsic excitability in neurons that stabilize cellular and neuronal network output is another mechanism that implies modified ion channel expression (Ransdell et al., 2012). Although 4-AP is blocking KV channels physically it has been found to evoke mRNA expression of KV channels in a feedback manner (Wang et al., 2012). Therefore it may be speculated that 4-AP induces the expression of ion channels that facilitates action potential conduction in demyelinated fibers.

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    Two authors contributed equally to this work.

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