Narahashi Ion Channels of Excitable Cells

1. Auflage 2013
ISBN: 978-1-4832-8833-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 19, 387 Seiten

Reihe: Methods in Neurosciences

Because of the highly significant and widely recognized roles of ion channels in physiology, pathophysiology, pharmacology, and toxicology, the term ion channel has now become a household word in the biomedical sciences. This volume covers preparations and techniques for the study of various ion channels. Both voltage-gated and ligand-gated ion channels of neurons, axons, and cardiac and smooth muscles are covered. It includes not only patch clamp techniques but molecular biology and imaging techniques as well. - Comprehensive protocols included for the study of: Ion channels using patch-clamp, molecular biology, and imaging techniques - Role of ion channels in physiology, pathophysiology, pharmacology, and toxicology - Specific ion channels of specific tissues

Autoren/Hrsg.

Narahashi, Toshio

Leseproben

1 Recording Sodium and Potassium Currents from Neuroblastoma Cells
Fred N. Quandt Publisher Summary
This chapter discusses the recording of sodium and potassium currents from neuroblastoma cells. Neuronal cell lines possess the functions of mammalian neurons in vivo, including electrical excitability. Neuroblastoma cells can grow under a wide variety of conditions. However, growth rates can vary. The growth rate can increase in proportion to the fetal bovine serum. The standard medium is Dulbecco’s modified Eagle medium. A bath perfusion system is employed to alter the electrolyte composition. A simple oscilloscope and pulse generator is used to measure the pipette and seal resistances. This chapter illustrates the feedback circuit that is used to minimize changes in the bath potential. The potential of the bath is measured with a low-resistance pipette filled with saline. An Ag/AgCl electrode in the bath is connected to the output of a negative feedback circuit to maintain the bath potential at ground. Single-channel recording can be obtained from membrane patches on intact neurons, inside-out, or outside-out patches. Introduction
Numerous studies of neuronal cell lines grown in tissue culture have shown that they possess the functions of mammalian neurons in vivo, including electrical excitability (McMorris et al., 1974). Voltage-clamp studies of neuroblastoma cells soon followed (Moolenaar and Spector, 1977, 1978, 1979; Kostyuk et al., 1978). These studies indicated that neuroblastoma cells would be a useful preparation for studying voltage-dependent Na and K channels (see also the review by Spector, 1981). I have often used N1E-115 neuroblastoma to examine the pharmacology of Na and K channels (Quandt and Narahashi, 1982; Quandt et al., 1985; Quandt, 1988b; Im and Quandt, 1992; Quandt and Im, 1992). The neurons have the advantage that they represent a convenient, homogeneous, and stable preparation for expression of these channels. Sodium and potassium currents can be easily measured from the whole cell, and single-channel analysis can be undertaken. In this article, I give the procedures which my laboratory has used for measuring these currents. Cell Culturing
Neuroblastoma cells will grow under a wide variety of conditions. However, growth rates will vary. It is convenient to plate 1 ml of cells into a 75-cm2 flask (Corning 25110-75) once a week, usually on Friday, and adjust conditions so that they become confluent in 7 days. When plated on Friday, the cells do not have to be fed until Monday. They are then fed every day until split on the next Friday. The growth rate appears to increase in proportion to the fetal bovine serum (FBS). We typically supplement the media with 5% FBS to maintain this growth rate. Standard medium is composed of Dulbecco’s modified Eagle’s medium (Sigma D5523 or GIBCO 430-1600). Glucose is brought to 4.5 g/liter by the addition of 3.5 g/liter. Sodium bicarbonate is added in the amount of 3.7 g/liter and 4.25 g/liter HEPES is also added in order to maintain pH in the CO2 incubator as well as in room air. We add 50 ml/liter of fetal bovine serum (Sigma F2138 or GIBCO 230-6140). Medium is then filtered with a 0.22 µm filter (Millipore Sterivex-GS, SVGSB1010) and stored in the refrigerator in 500-ml bottles. A total of 5 ml antibiotic-antimycotic solution (Sigma A9909 or GIBCO 600-5240) is added to an aliquot before its use. Cells grow well for more than 40 to 50 passages (in this case, corresponding to the number of weeks in culture). With the high passage numbers, we find that cells clump and do not extend processes when exposed to a differentiating condition (defined below). We then culture new cells which were previously frozen. Although we have no specific information to indicate that the electrophysiological properties of the cells are altered at the higher passage numbers, this procedure avoids potential variability. Our primary stock of cells is preserved at passage number 20 to 25, so that cells are thawed twice per year. To freeze cells, one flask is pelleted at 2000 rpm for 5 min and resuspended in 1 ml of growth medium to which dimethyl sulfoxide (DMSO) has been added at a concentration of 10% (v/v). This aliquot is then placed into a cryovial and placed into a styrofoam box. The box is placed into a -70°C freezer overnight. We find that this procedure gives a good rate of change of temperature for the freezing process. The vial is then stored in the liquid or vapor phase in a liquid nitrogen tank. Cells can be stored for many years. Cells are thawed by quickly bringing them to 37°C via immersion of the cryovial into a water bath. The thawed cells are transferred to a 15-ml centrifuge tube and 10 ml of normal growth medium is added slowly in order to avoid osmotic shock. The resuspended cells are then placed into a 75-cm2 flask. The solution should be changed the next day to remove the DMSO. Prior to their use in experiments, cells are grown on 22-mm-diameter glass coverslips (Fisher 12-546-1) in Dulbecco’s medium with 1 to 2.5% FBS and 1 to 2% DMSO. Three of the glass coverslips can be accommodated in 60-mm-diameter petri dishes (Falcon 1007). The coverslips are not routinely washed. The density of cells is not very critical, as long as the cells are not crowded. Cell growth is arrested on exposure to the altered medium, and many cells become differentiated after 3 days (see below). Recording Techniques
Numerous techniques have been used in this laboratory to record membrane potentials and currents from neuroblastoma cells. The two-microelectrode recording technique was the first method used to voltage-clamp neuroblastoma cells (see Introduction). The technique is not optimal because rather large microelectrodes must be used (5 to 10 MO when filled with 3 M KCl) due to the large currents. Under these conditions we have found that the stability of the clamp is limited to 5 to 10 min, since KCl leaks out of the electrodes, causing the cells to swell. The internal dialysis technique (Lee et al., 1980; Kostyuk et al., 1977) has been adapted to neuroblastoma cells (Kostyuk et al., 1978; Huang et al., 1982; Matsuki et al., 1983; Quandt and Narahashi, 1984). This technique uses a suction pipette with a large opening at the tip, with continuous perfusion of the inside, and has major advantages. First, the clamp is very stable, often allowing recordings for an hour. Second, the pipette resistance is low, minimizing series resistance. Third, it allows the solution to be changed during an experiment. Although large neurons are required, N1E-115 cells attain the required size. Use of the patch-clamp technique (Hamill et al., 1981) has superceded the suction pipette for measuring currents from the whole cell since it is more convenient and easier to switch to single-channel recording. Techniques are available to alter the internal pipette solution (Tang et al., 1990). The series resistance tends to be higher in the whole-cell patch clamp than with a suction pipette. On occasion I have substituted two patch electrodes for the microelectrodes in the two-electrode voltage clamp in order to eliminate the series resistance. It is also possible to establish a whole-cell patch clamp and simultaneously record current through a patch of membrane with an independent patch pipette. This last configuration eliminates the current through the seal resistance and reduces the capacitative current for the membrane patch. Whole-Cell Patch-Clamp Technique
Neuroblastoma cells can be readily patch-clamped following the general techniques which are given in Hamill et al. (1981). Whole-cell voltage clamp and single-channel recording from intact and excised membrane patches are rather routine. Recording System
A coverslip on which cells are grown is placed into a plastic chamber, sometimes on a small drop of Vaseline to prevent movement. The glass can first be broken using a sharp-tipped tool to reduce the size of the chamber and maintain the cells not being used. The chamber is fixed to the mechanical stage on an inverted microscope. The cells are visualized with the microscope using long working distance, phase contrast objectives and a total magnification of 400 to 600×. The microscope power supply can be replaced with a DC power supply to reduce 60-cycle noise in recordings. The patch pipette is maneuvered onto the cell using a Narashige three-dimensional hydraulic manipulator mounted on a second mechanical three-dimensional manipulator. The electrode is aimed at the cell at a 45° angle to the microscope stage. An anti-vibration air table will increase stability for whole-cell and intact patch recording situations. The controls for the hydraulic manipulator and the valve to the bath perfusion system are mounted off the air table to eliminate vibration following initiation of the clamp. We employ a bath perfusion system in order to alter the electrolyte composition or add neuroactive agents. In addition,...