Yonsei Med J.  2008 Jun;49(3):459-471. 10.3349/ymj.2008.49.3.459.

Changes in Inward Rectifier K+ Channels in Hepatic Stellate Cells During Primary Culture

Affiliations
  • 1Department of Physiology, College of Medicine, Pochon CHA University, Seongnam, Gyeonggi-do, Korea. qsang@yonsei.ac.kr
  • 2Department of Physiology and Institute of Lifelong Health, Yonsei University Wonju College of Medicine, Wonju, Gangwon-do, Korea.

Abstract

PURPOSE
This study examined the expression and function of inward rectifier K+ channels in cultured rat hepatic stellate cells (HSC). MATERIALS AND METHODS: The expression of inward rectifier K+ channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. RESULTS: The dominant inward rectifier K+ channel subtypes were K(ir)2.1 and K(ir)6.1. These dominant K+ channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K+ current (type 1) and the other without (type 2). The inward current was blocked by Ba2+ (100micrometer) and enhanced by high K+ (140mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba2+-sensitive current and the membrane potential. In addition, Ba2+ (300micrometer) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. CONCLUSION: HSC express inward rectifier K+ channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K+ channels in HSC as well as their roles in the activation process.

Keyword

Hepatic stellate cells; inward rectifier K+ channels; electrophysiology; real-time RT-PCR

MeSH Terms

Animals
Barium/pharmacology
Blotting, Western
Cells, Cultured
Electrophysiology
Liver/cytology/*metabolism
Male
Membrane Potentials/drug effects
Potassium/pharmacology
Potassium Channels, Inwardly Rectifying/genetics/metabolism/*physiology
Rats
Rats, Sprague-Dawley
Reverse Transcriptase Polymerase Chain Reaction

Figure

  • Fig. 1 Relative α-SMA and CaV1.2 gene expression in hepatic stellate cells (HSC). The dependence of α-SMA and CaV1.2 gene expression on activation was estimated using real-time RT-PCR. The HSC were used at 1 day, 1 week, 2 weeks, and 3 weeks of primary culture. The cells were prepared and processed as described in the Materials and Methods section. These time points represent the progressive transformation of quiescent HSC into activated HSC. The bar graphs show the relative α-SMA (A) and CaV1.2 (B) gene expression. Data were normalized to GAPDH and calibrated by α-SMA and CaV1.2 from HSC cultured for 1 day. The data are shown as the mean ± SEM (n = 3).

  • Fig. 2 Relative inward rectifier K+ channel and SUR gene expression in HSC. (A) The inward rectifier K+ channel α-subunit gene expression in HSC was measured using real-time RT-PCR. The bar graphs show the relative gene expression for each inward rectifier K+ channel subfamily (Kir1.1, Kir2.1 - Kir2.4, Kir3.1 - Kir3.4, Kir4.1 - Kir4.2, Kir5.1, Kir6.1 - Kir6.2, and Kir7.1). (B) SUR gene expression was measured. The bar graphs show the relative gene expression for SUR1, SUR2A, and SUR2B. The HSC were used at 1 day, 1 week, 2 weeks, and 3 weeks of culture. The expression levels were normalized to GAPDH and calibrated by Kir2.1 expression in the HSC cultured for 1 day. The data are shown as the mean ± SEM (n = 3). (C) Kir2.1 and Kir6.1 protein expression were measured by Western blotting. There was a band (55 kD) in the anti-Kir2.1 membrane (Upper) as well as the anti-Kir6.1 membrane (Lower). Both membranes were re-probed with anti-GAPDH antibody as shown. The data is representative of three independent experiments.

  • Fig. 3 Different whole-cell K+ currents in cultured HSC. The representative traces of K+ currents in types 1 and 2 HSC are illustrated in A and B, respectively. Voltage-dependent alterations in the membrane currents in a single HSC cultured for two days were measured under voltage-clamp conditions using the whole cell configuration of the gramicidin-perforated patch-clamp technique. The K+ currents were elicited by voltage ramps between - 140 to 60mV from a - 60mV holding potential. The representative current traces were measured before and after applying 100 µM Ba2+ (control-black, Ba2+-red). The I-V relationships of type 1 (C) and type 2 (D) were measured in high K+ (140 mM) and high K+ with Ba2+ (100 µM). Representative traces of the HSC cultured for three days are superimposed (control-black, high K+-blue, high K+ with Ba2+-red). All measurements were carried out with a physiological solution in the bath and the recording pipette filled with high K+ solution, as described in the Materials and Methods section.

  • Fig. 4 Relationship between resting membrane potential and Ba2+-sensitive current in HSC. (A) The relationship between the resting membrane potential and the Ba2+-sensitive current is shown (n = 81). The resting membrane potential was recorded from HSC under a current clamp. The graph was fitted by linear regression (r2 = 0.75). The dotted lines indicate the discriminative values for each parameter. (B) Comparison of the resting membrane potential in type 1 and 2 HSC (n = 52 and 60, respectively; p < 0.0001). (C-F) Changes in the membrane potential due to Ba2+ (300 µM) were measured in both types of HSC. The representative membrane potentials in HSC cultured for three days are shown in C (type 1) and D (type 2), and the bar graph shows the average of the control and Ba2+-treated membrane potentials in E (type 1) and F (type 2) (n = 16, 15, respectively). The data are reported as the mean ± SEM and *indicates p < 0.0001.

  • Fig. 5 Changes in the inward current and membrane potential in HSC during culture. (A) The amplitude of the inward current induced by high K+ (140 mM) from a holding potential of - 60 mV were examined as a function of culture time at 1 day, 1 week, and 2 weeks (Left panel: type 1, n = 26, 21, 22, respectively; Right panel: type 2, n = 23, 28, 29, respectively). The inward current, compensated for by membrane capacitance reflecting the membrane area, is presented as a current density (pA/pF). (B) The membrane potential measured in type 1 HSC is presented as three groups; HSC cultured for 1 day, 1 week, and 2 weeks (n = 20, 17, 19, respectively). The data are reported as the mean ± SEM, and * and ** indicate p < 0.05 and p < 0.001, respectively.


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