Intracellular electrical measurements

The automatic measurement of the extracellular and intracellular electrical potential difference can be effectively used in plant electrophysiology for studying the molecular interfacial mechanisms of ion transport, the influence of external stimuli on plants, and for investigating the bioelectrochemical aspects of the interaction between insects and plants. 

A heart muscle cell (rabbit cardiomyocyte) was aligned in a narrow PDMS-glass microchannel. The muscle cell was electrically stimulated via a pair of Au electrodes ( 60 pm apart) in contact with the cell. Intracellular Ca measurement showed that the cell remained contracted for 60 min within the restricted space. An electric field strength of 20 V/cm translates to 0.12 V, which is lower than the electrolysis threshold of 0.8 V [834]. 

The use of dielectric spectroscopy for the characterization of living cells and the possible derivation of cellular parameters such as living ceU volume concentration (Figure 4.14), complex permittivity of extracellular and intracellular media, and morphological factors is discussed by Gheorghiu (1996). Another possible application is the electrical measurement of erythrocyte deformability (Amoussou-Guenou et al., 1995). 

Electrical measurements on cells are discussed in other entries in the encyclopedia Patch Clamp Measurements On-CThip. The focus here is on the basic cell electrical responses assayed in microchannels, most of which have been developed in the context of cell-based biosensors. These studies typically evaluate cell action potential which is a net electrical change in cells resulting from changing concentrations of intracellular ions. Action potentials are important physiologically because they result in the... 

Figure 2 The principle of measurement by means of an ion-selective double-barreled microelectrode inside a cell. The cell is in a bath the solution of which is grounded via a reference electrode. Each barrel is connected via a chlorided silver wire (shown coiled) to amplifiers (triangles). The reference barrel of the double-barreled electrode directly records the intracellular electrical potential, the membrane potential (Em). The ion-selective barrel, indicated by the plug of ion exchanger in the tip, records the sum of the membrane potential and a potential , related to the chemical potential of the ion in question (of activity a) (see eqn [1]). / is obtained by electronic subtraction. The influence on from other ions (indicated by index j and the valencies Zy) can be obtained from calibration.

FIG. 2 Measurements of intracellular potentials in sieve tubes of maize via severed aphid stylets. Stimulation by ice water (above) and electric shock (below) evoked action potentials which were propagated with a velocity of 3-5cms in a basipetal direction. (From Ref. 36.)... 

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

Burdyga T, Wray S 1997 Simultaneous measurements of electrical activity, intracellular [Ca2+] and force in intact smooth muscle. Pfliigers Arch 435 182-184 Burdyga TV, Wray S 1999a The relationship between the action potential, intracellular calcium and force in intact phasic, guinea-pig uretic smooth muscle. J Physiol 520 867-883... 

Schematic representation of the heart and normal cardiac electrical activity (intracellular recordings from areas indicated and ECG). Sinoatrial (SA) node, atrioventricular (AV) node, and Purkinje cells display pacemaker activity (phase 4 depolarization). The ECG is the body surface manifestation of the depolarization and repolarization waves of the heart. The P wave is generated by atrial depolarization, the QRS by ventricular muscle depolarization, and the T wave by ventricular repolarization. Thus, the PR interval is a measure of conduction time from atrium to ventricle, and the QRS duration indicates the time required for all of the ventricular cells to be activated (ie, the intraventricular conduction time). The QT interval reflects the duration of the ventricular action potential.

For each of the above protocols paired measurements of one or several given parameters of tubule transport are obtained under control conditions and in the presence of a substance under study. Also concentration response curves can be obtained in one single preparation (Schlatter et al. 1983 Wangemann et al. 1986 Wittner et al. 1987). Intracellular measurements are usually required to define the mechanism of action (Greger 1985). Especially the electrical and optical measurements have a very high reproducibility. For screening usually 3 preparations are sufficient. Approximately 10 preparations are required for concentration response curves. 

In this table, P represents anions of protein and organic phosphate. The membrane is permeable to the group represented by P. The mean values of the charge on P are -6.7 and -1.08 for the interior and the exterior of the cell, respectively. An electrical potential difference of At// = i/t, t// = 90 mV is measured, i and o denote the intracellular and extracellular, respectively. The activity coefficients of components inside and outside the cell are assumed to be the same, and pressure and temperature are 1 atm and 310 K. Assume that the diffusion flows in from the surroundings are positive and the diffusion flows out are negative. Using tracers, the unidirectional flows are determined as follows ... 

FLIM measurements were applied to Hb. salinarum loaded with BCECF. At least two halobacteria species that exhibit different fluorescence lifetimes from each other are found to exist in the cell suspension, suggesting that the cells have different intracellular environments. The difference in the fluorescence lifetime may reflect the difference in activity of halobacteria. It is suggested that strong electric fields inside a cell play a significant role in the determination of the fluorescence lifetime of BC ECF. 

Electrical properties of neurons are measured by a variety of techniques. A review of these methodologies is beyond the purview of this chapter. However, it is important to note that changes in the electrochemical potential, ionic diffusion or current, and membrane conductance or permeability can be determined experimentally by intracellular and extracellular recording techniques that can be performed in vitro and in vivo. Hubbard et al. (1969) describe in detail a variety of intracellular techniques, such as voltage clamping, and extracellular techniques, such as sucrose-gap recording. 

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