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MEHR ERFAHREN

VroniPlag Wiki


Typus
KomplettPlagiat
Bearbeiter
Hindemith
Gesichtet
No.png
Untersuchte Arbeit:
Seite: 8, Zeilen: 1ff (entire page)
Quelle: Embark 2004
Seite(n): 30, 31, 32, Zeilen: 30: 19ff; 31: 1ff; 32; 1ff
For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. While the nucleus resides in the animal pole [Nieuwkoop, 1977], more mRNA is present in the vegetable pole [Capco and Jeffery, 1982]. The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, [Dascal, 1987].

The primary advantage of using Xenopus oocytes for the expression of transporters is the ability to perform detailed electrophysiological recording using an in vivo system. In the simplest arrangement, the membrane is penetrated with a single microelectrode and the membrane potential is measured. The oocyte can easily be penetrated with two microelectrodes. This arrangement allows the use of one of the two classical methods: current clamp or voltage clamp. Most electrophysiological studies on oocytes were performed using the two-electrode voltage-clamp. The large size of the oocytes also permits extracellular recording of currents flowing through the cell membrane at various locations using a vibrating probe. The patch clamp method has been successfully applied in devitellinized oocytes for the study of single channels [Hamill et al., 1981].

Whole-cell voltage clamping of oocytes involves two electrodes inserted into the oocyte. The large size of the oocyte (about 1 mm in diameter and 0.5 to 1 μl in volume for stage V-VI oocytes) make this feasible, and is both the major advantage and disadvantage of the system. The advantage is that it is possible to insert multiple electrodes and injection needles into the same oocyte. Therefore, modulators of channel function can be injected inside the cell while recording, so that a rapid and direct response to an intracellular signal can be observed. The disadvantage is that the large size results in an extremely large membrane capacitance (about 150-200 nF), which causes a slow clamp setting time following voltage shifts. This makes it difficult to obtain any data during the first 1 to 2 msec of a hyper- or depolarization, the time during which rapidly activating voltage sensitive channels such as the cardiac sodium channel open. The large capacitance is not a serious problem in examining slow responses or ligand-gated responses in the absence of voltage shifts [Stuhmer, 1992]. Despite their advantages, several precautions should be taken into consideration.

Firstly, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high level can induce endogenous channels. [Tzounopoulos et al.,1995].

Secondly, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temprature & most experiments are carried out at room teperature. Hence, tempreture sensitive processes i.e. protein trafficking or kinethics may be altered.

Finally, since Xenopus oocytes may have different signaling pathways, precatuion should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA & PKC pathway. However, in Napi-3 expressing Xenopus oocytes PKC-mediated PTH regulation can not be observed [Wagner et al.,1996]. Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels [Waldegger et al.,1996].

For experimental studies oocytes of stages V-VI are used with a diameter of some 1.3 mm allowing easy preparation. The developmental stages V-VI are characterized by the occurence of 2 poles i.e. the vegetable (light) and the animal (dark) poles. While the nucleus resides in the animal pole (Nieuwkoop, 1977), more mRNA is present in the vegetable pole (Capco and Jeffery, 1982). The main ion conductance in Xenopus oocytes is a Ca2+-dependent Cl- conductance governing the resting membrane potential close to the Cl- reversal potential of -40 mV, (Dascal, 1987).

The primary advantage of using Xenopus oocytes for the expression of transporters is the ability to perform detailed electrophysiological recording using an in vivo system. In the simplest arrangement, the membrane is penetrated with a single microelectrode and the membrane potential is measured. The oocyte can easily be penetrated with two microelectrodes. This

[page 31]

arrangement allows the use of one of the two classical methods: current clamp or voltage clamp. Most electrophysiological studies on oocytes were performed using the two-electrode voltage-clamp. The large size of the oocytes also permits extracellular recording of currents flowing through the cell membrane at various locations using a vibrating probe. The patch clamp method has been successfully applied in devitellinized oocytes for the study of single channels (Hamill et al., 1981).

Whole-cell voltage clamping of oocytes involves two electrodes inserted into the oocyte. The large size of the oocyte (about 1 mm in diameter and 0.5 to 1 μl in volume for stage V-VI oocytes) make this feasible, and is both the major advantage and disadvantage of the system. The advantage is that it is possible to insert multiple electrodes and injection needles into the same oocyte. Therefore, modulators of channel function can be injected inside the cell while recording, so that a rapid and direct response to an intracellular signal can be observed. The disadvantage is that the large size results in an extremely large membrane capacitance (about 150-200 nF), which causes a slow clamp setting time following voltage shifts. This makes it difficult to obtain any data during the first 1 to 2 msec of a hyper- or depolarization, the time during which rapidly activating voltage sensitive channels such as the cardiac sodium channel open. The large capacitance is not a serious problem in examining slow responses or ligand-gated responses in the absence of voltage shifts (Stuhmer, 1992).

Despite their advantages, several precautions should be taken into consideration. First, the expression of endogenous carriers may interfere with the exogenously expressed proteins in various ways. For instance, it has been observed that injection of heterologous membrane proteins at high levels can induce endogenous channels (Tzounopoulos et al., 1995). Second, due to the fact that Xenopus laevis is a poikilothermic animal, its oocytes are best kept at lower temperature and most experiments are carried out at room temperature. Hence, temperature sensitive processes i.e. protein trafficking or kinetics may be altered (Wagner et al., 2000).

[page 32]

Finally, since Xenopus oocytes may have different signaling pathways, precaution should be taken when studying the regulation of expressed proteins. It has been revealed that the PTH receptor regulates the internalization of NaPi, mediated by the PKA and PKC pathway. However, in NaPi-3 expressing Xenopus oocytes PKC-mediated PTH regulation can not be observed (Wagner et al., 1996). Instead, coupling to the PKA pathway leads to the alteration of PKA-regulated ion channels (Waldegger et al., 1996).

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(Hindemith)