The plasma membrane of a cell typically has a transmembrane potential of approximately -70 mV (negative inside) as a consequence of K⁺, Na⁺ and Cl^– concentration gradients that are maintained by active transport processes. Increases and decreases in membrane potential (referred to as membrane hyperpolarization and depolarization, respectively) play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling, and ion-channel gating.

Potentiometric optical probes enable researchers to perform membrane potential measurements in organelles and in cells that are too small to allow the use of microelectrodes such as the patch clamp technique. Moreover, in conjunction with imaging techniques, these probes can be employed to map variations in membrane potential across excitable cells and perfused organs with spatial resolution and sampling frequency that are difficult to achieve using microelectrodes.

Potentiometric probes are important tools for studying these processes, as well as for visualizing mitochondria (which exhibit transmembrane potentials of approximately -150 mV, negative inside matrix), cell-viability assessment, and high-throughput screening for new drug candidates. Potentiometric probes include: the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic oxonols and hybrid oxonols, and merocyanine 540.

Fluorescent indicators of membrane potential have been broadly classified as either slow (redistribution, or nernstian) or fast (electrochromic) dyes. There is a fundamental difference between these two distinct families of dyes with regard to the mechanism of their response to membrane potential, speed of the response, and the range of cellular characteristics.

Fast-response probes (usually styrylpyridinium dyes) operate by means of a change in their electronic structure, and consequently their fluorescence properties, in response to a change in the surrounding electric field. Their optical response is sufficiently fast to detect transient (millisecond) potential changes in excitable cells, including single neurons, cardiac cells, and intact brains. However, the magnitude of their potential-dependent fluorescence change is often small; fast-response probes typically show a 2-10% fluorescence change per 100 mV.

Slow-response probes exhibit potential-dependent changes in their transmembrane distribution that are accompanied by a fluorescence change. The magnitude of their optical responses is much larger than that of fast-response probes (typically a 1% fluorescence change per mV). Slow-response probes, which include cationic carbocyanines and rhodamines and anionic oxonols, are suitable for detecting changes in average membrane potentials of nonexcitable cells caused by respiratory activity, ion-channel permeability, drug binding, and other factors.

A widely used fluorescent dye for measuring transmembrane potential, DiSBAC2(3), has a limitation of poor dynamic range in an assay environment in the absence of serum. AnaSpec has developed a novel membrane potential-sensitive fluorescent dye, HLB021-152, and successfully used it for homogeneous live-cell cAMP assay in both serum-containing and serum-free environment. Upon stimulating the endogenous or heterogenous GPCRs on CNG-channel-cloned HEK 293 cells with agonists, the fluorescent signal of HLB021-152 increased rapidly and had greater dynamic range than DiSBAC2(3). This new membrane potential-sensitive dye can be formulated for high throughput screening of GPCR modulators in both with serum and without serum environments (Assay Drug Dev Technol. 2006 Aug; 4(4): 461-71).

Figure 1. Extra and intra-cellular distribution of HLB021-152 before and after the addition of KCl. 3T3 was incubated with HLB021-152 (30 mM) and a quencher solution for 30 min. After which 120 mM KCl was added to depolarize the membrane. A. Fluorescent image before the addition of KCl. B. Fluorescent image at 30^th sec after the addition of 120 mM KCl. The filter set with exciter at 545/30 nm and emitter at 610/75 nm was used. Several fields had been examined and showed consistent results.

Figure 2. Comparison of the dynamic range of HLB021-152 and DiSBAC2(3) in measuring the dose response of 5-hydroxytryptamine (5-HT) in HTR7B-CNG cell line. HTR7B-CNG cells cultured in serum-free medium was loaded with HLB021-152 (30mM) or DiSBAC2(3) (30 mM) dye/quencher solution for 2hr. Serially diluted 5-HT was added into the cells to stimulate human 5-hydroxytryptamine receptor 7B. Fluorescence signals of HLB021-152 and DiSBAC2(3) were recorded before adding 5-HT (F₀) or 30 minutes after adding 5-HT (F). F/F₀ was calculated and plotted versus the concentration of 5-HT. (n=4, mean±SD)

AnaSpec is pleased to highlight the fast- and slow-response probes for fluorimetric measurememt of membrane potentials. These fluorescent reagents are of the highest quality, and are cost-effective.