Monitoring Ion Activities In and Around Cells Using Ion-Selective Liquid-Membrane Microelectrodes

Determining the effective concentration (i.e., activity) of ions in and around living cells is important to our understanding of the contribution of those ions to cellular function. Moreover, monitoring changes in ion activities in and around cells is informative about the actions of the transporters and/or channels operating in the cell membrane. The activity of an ion can be measured using a glass microelectrode that includes in its tip a liquid-membrane doped with an ion-selective ionophore. Because these electrodes can be fabricated with tip diameters that are less than 1 μm, they can be used to impale single cells in order to monitor the activities of intracellular ions. This review summarizes the history, theory, and practice of ion-selective microelectrode use and brings together a number of classic and recent examples of their usefulness in the realm of physiological study.


Introduction
The activity of an ion is the effective concentration of that ion in a mixture of chemicals. This value is generally slightly smaller than the molar concentration of the parent chemical species because some salts do not fully ionize in solution. Moreover, the extent of ionization can be influenced by the presence of other chemical species in the solution as well as by factors such as temperature and ionic strength. Importantly it is the activity (a), rather than the concentration, of an ion that determines the OPEN ACCESS thermodynamic contribution of the ion to a system such as membrane potential (a parameter that is predominantly determined by the intracellular and extracellular activities of Na + , K + , and Cl − ) and the rate of chemical reactions in which physiologists are interested. Every cell has the ability to control the distribution of ions across its membranes by virtue of the channels and transporters that are present in each membrane.
Three classical technologies that are applied to determining ion activity/concentration and monitoring the movement of ions across cell membranes are radiolabeled tracers (e.g., reference [1]), ion-sensitive fluorescent indicator dyes [2], and ion-selective microelectrodes (ISMs). ISMs based on ion-selective liquid-membranes are the focus of the present review. These ISMs are glass microelectrodes that are used to continuously monitor the activity of a specific ion at a specific locus by virtue of their tips being filled with an ion-selective liquid membrane. ISM use facilitates ion measurement because 1. Numerous ISMs can be applied to a single cell at the same time, allowing numerous ion activities to be monitored simultaneously. 2. ISMs can be applied to monitor ion activity at specific loci such as the cell surface or the cytoplasm. 3. The reference electrode that is paired with the ISM for measurement of intracellular ion activity (see Section 5) provides a simultaneous measurement of membrane potential providing a more complete characterization of the transport processes that contribute to the changes in ion activities. 4. In combination with vibrating probe technology [3], ISMs can be used to measure net ion fluxes.
2. The use of ISMs to monitor intracellular ion activities is best applied to large cells that can be easily impaled with a microelectrode (e.g., Xenopus oocytes, which have a diameter that is greater than 1 mm; approximately 50-100 times larger than a typical mammalian cell).
The earliest ISMs were fabricated from ion-selective glass [4] but their usefulness for intracellular ion-measurements, which requires impalement of a cell, is limited by their relatively large tip diameter, slow response time, and expertise required to fabricate them [5]. However their worth is evidenced by studies of, for example, pH i regulation in snail neurons [6], giant-barnacle muscle-fibers [7], and squid axons [8] that were conducted using ISMs based on H + -selective glass.
The replacement of an ion-selective glass tip with an ionophore-doped liquid membrane (also known as an ionophore cocktail) conferred ion selectivity to glass microelectrodes with a smaller (less than 1 μm) diameter tip and a t 90 -the time taken for 90% of the full electrode response to occur-on the order of seconds (see reference [9] for a review of more recently recommended measures of ion-selective electrode response times). Here, we discuss the theory, fabrication, and application of ionophore-cocktail based ISMs. The theory, construction, and application of ISMs have also been reviewed by others in references [4,[10][11][12][13].

Theory of ISMs
In order for an ISM to be useful, the microelectrode must respond predictably and rapidly to changes in ion activity such that the voltage reported by the microelectrode can be used to recreate information about ion concentration. Underlying the theoretical considerations that describe the relationship between the electrical signal reported by an ISM and the ionic composition of the solution to which the ISM is exposed are the concepts of ion activity and electrochemical potential. In the following subsections, we first consider the theoretical behavior of gases in a closed system and extend that theory to uncharged solutes and ions. We then consider the distribution of a single ion species across a semi-permeable membrane, and finally the electrochemical potential difference across an ion-selective liquid membrane such as an ionophore cocktail.

Gibbs Energy
Gibbs energy (G) is the potential energy that can be absorbed or released during a chemical reaction in a closed system. G is a function of internal energy (U), pressure (P), volume (V), temperature (T), and entropy (S) as shown in Equation (1): Equation (1) can be differentiated to: (2) Equation (2) can be simplified because (the first law of thermodynamics, in which Q is heat) and (the second law of thermodynamics). Thus at fixed temperature ( ), Equation (2) can be restated as: Substituting the definition of into Equation (3) according to the ideal gas law (  ),  where is the ideal gas constant, describes the relationship between G and P: is defined as an infinitesimally small change in G in Equations (2), (3), and (4). But if we assume that our model system changes from an initial state "1" to a final state "2", causing a measureable change in G ( ), we can integrate Equation (4) between states 1 2, as shown below in Equation (5). (Note that ): That is to say: Defining the initial state 1 as a standard state where G ⊖ is the standard free energy and P ⊖ is the standard state pressure (1 atm), provides us with Equation (7): .
In our closed system (at constant T, V) Equation (7) can be restated in terms of chemical potential , which can be described as Gibbs energy per mole ( ):

Chemical Potential and Activity
In the case of a solution, it is more useful to consider the concentration of a molecule ([X]) rather than its pressure ( P ). Henry's law relates pressure and concentration by a constant k H that cancels out when we substitute the equation into Equation (8): Because the standard state concentration of particle , this term does not need to be included in the equation. As we noted earlier, because of the influence of physical factors upon solubilization, the activity of a chemical species ( ) is usually less than its molar concentration [14]. Thus we need to modify the relationship with an activity co-efficient ( ) which relates the activity and concentration of a species ( ) such that:

Electrochemical Potential
In order to extend our consideration to charged particles (i.e., ions) we must further modify Equation (10) to describe the contribution of charge to the electrochemical potential of the system, where z is the valence of the ion, F is Faraday's constant, and is electrostatic potential of the system: (11) Note that, if a particle is uncharged, and , per Equation (10).

Electrochemical Potential Difference across a Semi-Permeable Membrane
Now, let us imagine two compartments 1 and 2 that are separated by a selectively-permeable barrier such as a cell membrane or an ionophore cocktail that permits selective permeation of a certain ion (shown in Figure 1, panels A and B). That is to say, when both compartments contain an equal concentration of , (Figure 1(A)) but a ten-fold difference in across an -permeable membrane will produce an electrical potential difference across that membrane of 0.058 V = 58 mV in the case of a monovalent ion (Figure 1(B)), or 29 mV in the case of a divalent ion. This predictable electrical response to changes in ion activity is the basis for the usefulness of ISMs.

Electrochemical Potential Difference across an Ion-Selective Liquid Membrane
In the case of an ISM, we can consider the ionophore cocktail in the tip of the electrode as the semi-permeable membrane. A 'backfill' solution within the ISM (Figure 1(C); equivalent to compartment 2 in Figures 1(A) and 1(B)) provides the electrical connection between a silver wire (Ag/AgCl half-cell) in the microelectrode holder (see Section 5.1) and the ionophore cocktail. The assay space into which the electrode tip is placed, of which we would like to know the ion concentration ( Figure 1(C)), is equivalent to compartment 1 in Figures 1(A) and 1(B). Thus a ten-fold concentration difference between in the assay space and in the backfill solution is registered as a mV potential difference across the cocktail. Crucially, given that the backfill solution in compartment 2 has a fixed composition ( ) and assuming that the activity co-efficient for is constant in the assay space, we can determine that the potential difference between two solutions A and B containing and is: That is to say, a ten-fold concentration difference between solution A and solution B will register as a 58 mV potential difference between the two solutions for a monovalent ion [ Figure 1(D)]: a Nernstian response. The exhibition of a Nernstian response by an ISM proves that it is selectively permeable to the ion of interest among all ions present in the solutions. This is the basis of calibration, an example of which is shown in Figure 2. Practical aspects of electrode calibration are considered in Section 5.3. Traditionally, K + -and H + -selective ISMs are more ideally ion-selective than Na + -or Cl − -selective ISMs. A consideration of the selectivity of each cocktail is presented in Sections 3.1 to 3.4 (guidelines for determining the selectivity coefficients for ISMs are provided in reference [15]). Note that the activity of Na + decreases as [Na + ] increases causing the calibration slope of Na + -selective ISM in Figure 2 to be slightly less than the Nernstian ideal of 58 mV/decade. Note that the estimated activity coefficients of the ions considered in this review (H + , Na + , K + , and Cl − ), are 0.98 at 1 mM, 0.93 at 10 mM, and 0.82-0.86 at 100 mM [14].

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There a microelectro onophore I/ All three co physiologica ncorporatin pH 4.5) whi pH 2.0 not a Ionophore I is tridodecylamine (TDDA), a lipophilic amine that acts as a proton carrier. In our laboratory we routinely use ISMs containing Ionophore I/Cocktail B (see Table 1) to monitor intracellular and extracellular pH. Cocktails A and B differ only in the identity of the cocktail additive inasmuch as Cocktail A contains sodium tetraphenylborate (NaTPB) rather than potassium tetrakis (4-chlorophenyl)borate (KTCPB). Both cocktails behave essentially the same, both being extremely selective for H + over other ions such as Na + in the physiological range (greater than 10 12 -fold preference) and both exhibiting similar response times. The authors who first reported the use of ISMs based on TDDA recommended overnight incubation of cocktail A with 100% CO 2 prior to use [16]. This procedure was presumably a precaution to minimize drift of potential due to CO 2 interference when the ISM is used in biological systems. However, CO 2 does not interfere appreciably with Cocktail B due to the substitution of the NaTPB additive with KTCPB [12]. Notably, a modified ISM that is fabricated with a CO 2 -permeable column of Ionophore I and a backfill that contains carbonic anhydrase can be used in concert with a H + -selective ISM to monitor pCO 2 [17].
Ionophore II/Cocktail A is composed of the same solvent and additive as Ionophore I/Cocktail B, albeit in a slightly different ratio, but is based on a different H + ionophore, namely 4-nonadecylpyridine (CAS No. 70268-36-9), which has a slightly poorer cation selectivity.

K + -Selective Ionophore Cocktails
There are two commercially-available cocktails recommended for use in K + -selective microelectrodes: Ionophore I/Cocktail A (cat. no. 60031; Sigma, see Table 3) and Ionophore I/Cocktail B (cat. no. 60398; Sigma). Ionophore I is valinomycin, which carries K + across membranes [21]. Both cocktails are similar in composition but cocktail A contains the solvent dibutyl sebacate, which confers upon the cocktail an improved selectivity for K + over Na + .

A Cl − -Selective Ionophore Cocktail
At the time of writing, there is only one commercially available cocktail recommended for use in Cl − -selective microelectrodes: Ionophore I/Cocktail A (cat. no. 99408; Sigma, see Table 4). This cocktail is about 30-fold more selective for Cl − over HCO 3 − [25], but is considered poorly selective with regard to its target ion compared to ion-selective cocktails for H + , Na + , and K + . The presence of HCO 3 − at close-to-physiological concentration does not substantially interfere with the Nernstian response of the electrode to Cl − , but if the electrode is to be used in the presence of HCO 3 − , it should be calibrated in the presence of HCO 3 − in order to correct for interference [26]. Great care should be taken to account for interference by HCO 3 − when designing and interpreting the results of experiments that employ this cocktail, especially (1) if used in an assay space with an alkaline pH (above pH 7.6, see reference [25]), (2) if used in compartments such as the cytoplasm that could contain interfering anions that are beyond the investigators control, or (3)

if [HCO 3
− ] is expected to change substantially over the assay period.

Pulling Glass Microelectrodes from Capillary Glass
Glass microelectrode can be fabricated with either a sharp end (tip diameter~1 μm) for intracellular recordings or a 'blunt' end (tip diameter~20 μm) for cell-surface/extracellular recordings. We pull our microelectrodes from borosilicate capillary glass using a micropipette puller (for example, we use a Model P-97 Flaming/Brown from Sutter Instrument, Novato, CA, USA). An example of each type of electrode is shown in Figure 3.

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3, 13
in Equation potential b n B is ext ded microel order to ca mobilities, j hen the refe true ion-sel differences ale a cell fo that doe otential diffe alibrated). T in Figure 5   reference electrode that senses the electrical potential of the assay space. Both probes should be mounted onto micromanipulators to allow fine positioning of the electrode tips. Three features of the electrical set up allow accurate monitoring of the voltage signal from each electrode. Firstly, the amplifiers have an input-resistance/impedance ( > 10 15 Ω) that is many times greater than that of the electrode tip (for sharp-ended ISMs, is typically ~10 11 Ω). This is an important consideration because the current that relays the input voltage signal ( ) from the assay space passes through the ISM tip as well as the amplifier and thus is influenced by the sum of their impedances ( ), whereas the current that relays the output voltage signal ( ) is influenced only by the impedance of the amplifier ( ). A restatement of Ohm's law relates these quantities as: (19) Thus, the relatively high impedance of the amplifier compared to that of the ISM tip minimizes the loss of signal amplitude by ensuring that both and are influenced by total impedances of similar magnitude. Furthermore the higher impedance of the amplifier reduces phase shift which could distort the relationship between and . Secondly, a signal-driven shield built into each probe, feeds a duplicate signal into the cable shielding, a maneuver that prevents stray capacitance between the signal-carrying wire and cable-shielding, increasing the responsiveness of the electrode. The shield also reduces electrical noise.
Thirdly, the dual-channel electrometer generates an ion-selective potential signal by subtracting the reference electrode signal ( ) from the ISM signal ( ) with a high common-mode rejection ratio, which effectively eliminates noise common to both channels such that the true differential (i.e., ion-selective) signal is accurately obtained, as depicted in Figure 5.
Note that, for intracellular recordings, the reference-electrode signal can be used to monitor membrane potential ( ) by clamping the electrical potential of the recording chamber to 0 mV using voltage-clamp circuitry (e.g., the OC-725C Oocyte Clamp from Warner Instruments), in which case . Alternatively the potential of the extracellular space ( ) can be monitored with a calomel electrode or a second saturated-KCl-filled microelectrode (connected to a separate amplifier) and subtracted from using a separate subtraction amplifier, in which case . Note that when the reference electrode is not impaled into a cell.

Calibration Procedure
The theory that underlies the calibration of ISMs is presented in Sections 2.5 and 5.2. An example calibration plot for a Na + -selective microelectrode is presented in Figure 2. A calibration slope of 58 mV/decade tells us that the ISM is ideally ion-selective with respect to its target ion. We use in-house software to convert the dual-channel electrometer output into a measure of ion concentration.
The series of solutions used to calibrate an ISM should have a composition that is close to that of the experimental sample to be measured (including potentially interfering ions), should cover the entire range of values expected to be encountered during the experiment, and should differ only in the concentration of target ion. For example, for calibration of a H + -selective ISM, we use a pH 6 buffer, a 223 FD pH 7.5 buffer, and a pH 8 buffer. For ISMs that are less-ideally ion selective, such as Na + -selective microelectrodes, more calibration points may be necessary (e.g., a five-point calibration is shown in Figure 2).

Applications of ISMs
ISMs are suitable for obtaining both intracellular and extracellular measurements and can be used to monitor ion activities at a specific locus or, by virtue of self-referencing/vibrating probe technology (reviewed in references [3] and [32]), between two loci in order to gather information about ion gradients and fluxes. In this section we briefly review a selection of physiological studies that demonstrate the usefulness of ISMs.
The original application of the H + -selective (ionophore-based) ISMs reported by Ammann and coworkers was determination of the intracellular pH (pH i ) of a Xenopus (frog) oocyte [16]. Cicirelli and coworkers extended this application to the study of changes in oocyte pH i during oocyte maturation [33]. Others have harnessed H + -selective ISMs to monitor the activities of heterologouslyexpressed H + -coupled transporters in oocytes. For example, these studies have been critical to understanding the molecular physiology of the monocarboxylate (H/lactate) cotransporter MCT1 [34] and the H + -coupled oligopeptide transporter PepT1 [35]. Of course, the same usefulness applies to any other ion-selective ISM, for example the use of a Na + -selective ISM to characterize a Na + -coupled transporter. The observation of ion-movement across the plasma membrane is critical to distinguish ion-transport from ion-dependence (e.g., a demonstration of increased lactate transport by MCT1 at low extracellular pH is not the same as a demonstration that H + is cotransported with lactate) and net ion-transport from gross ion-transport (e.g., see reference [26]).
H + -selective ISMs are also routinely used to monitor the other processes that affect the pH i of cells. For example, the action of the electrogenic Na/HCO 3 cotransporter NBCe1 (because HCO 3 − uptake consumes H + and raises pH i [36]), the movement of CO 2 across a cell membrane (because CO 2 is hydrated as it enters the cell, generating H + and lowering pH i [37]), the action of the NH 4 + /NH 3 channel AmtB [38], and the action of carbonic anhydrase II (that catalyzes the reversible hydration of CO 2 [37]). Because the measured signals are robust, H + -selective ISMs can also be applied to the comparison of the activities of wild-type and mutant proteins expressed in oocytes. For example, ISMs have been used by multiple groups to study the molecular defects in Na + and HCO 3 − transport by disease-associated mutants of human NBCe1 [39][40][41]. ISMs can also be applied to measure changes in extracellular ion activities around cells and in intact tissues (e.g., those associated with swelling activated channels in epithelial cells [42] or neuronal activity in hippocampal slices from rats [43,44]), and changes in extracellular ion activities in the brains of whole animals (e.g., anaesthetized rats [45] and flies [46]). The study of intracellular ion activities in cells that may only have a diameter of 10 μm, requires the use of smaller tip-diameter ISMs, with t 90 values that are on the order of tens of seconds. Because it is technically difficult to impale a small cell with both an ISM and a reference electrode, these studies are typically performed with double-barreled electrodes that combine the ISM and reference in a single tip. These have been used to monitor changes in intracellular ion activities in cells of isolated perfused tissues such as rabbit proximal tubules [25,47,48], sheep cardiac Purkinje fibers [49], and insect Malpighian tubule cells [50].
In the extracellular milieu, small changes in ion activity that result from ion channel or transporter activity can be difficult to reliably detect because local ion gradients at the transport site are quickly dissipated, especially if the cells are being superfused. In order to facilitate these measurements, investigators have developed the ion-trap technique [51][52][53] that uses a blunt-tipped ISM (Section 4.1) pushed up against the surface of a cell in order to isolate a small volume between the transport site and the ionophore cocktail within which a small accumulation or depletion of an ion will produce a substantial change in ion activity. This technique has been applied to the monitoring of CO 2 and NH 3 movement across the oocyte plasma membrane [54], the export of Cl − by anion exchangers in the presence of HCO 3 − [26], and the influx of H + mediated by the Na + /glucose cotransport SGLT1 in the absence of Na + [55]. As mentioned earlier, ISMs-with the exception of those based on H + -selective ionophores-are not overwhelmingly selective towards their intended target ion. However, this phenomenon can be used to an investigator's advantage. For example, the poor selectivity of some primitive "K + -selective" ISMs with respect to quaternary ammonium ions (such as the cell-impermeant cation tetramethylammonium, TMA + ) means that these ISMs can be exploited to monitor cell volume regulation in cells loaded with TMA + [56]. Underlying this application is the assumption that, if the intracellular TMA + content is fixed, changes in measured TMA + activity are due to changes in cell water content [56]. Another example of an "off-target' use of an ISM relates to the "Na + -selective" ISM discussed in Section 3.2. These Na + -selective ISMs are actually somewhat more selective for Li + than Na + . Because Li + is not a major component of physiological solutions, Li + interference with the ISM is not a usual consideration. However, in experiments conducted in the presence of extracellular Li + and absence of extracellular Na + an intracellular "Na + -selective" ISM can be used to monitor Li + import across the plasma membrane, assuming that intracellular Na + activity remains constant [57].

Outlook
The application of ISMs to the measurement of ion activities remains limited by the small size of individual cells in relation to the diameter of the ISM tip, which is why ISMs lend themselves better to use in large cells or tissue slices. Studies of ion perturbations in small, individual cells predominantly rely on the use of ion-sensitive dyes. In order to overcome size limitation, exciting efforts are currently underway to create miniaturized ISMs, and arrays of ISMs, by harnessing microfluidics technology (reviewed in reference [58]). Furthermore, ion-selective cocktails with novel and improved characteristics and ion-selectivities are constantly in development (e.g., [59][60][61] and review of ion-selective electrodes based on PVC-membranes that could be incorporated into ISMs in reference [62]), broadening the range of studies to which ISMs can be applied.