Mag-Fura-2 (Furaptra) Exhibits both Low (µM) and High (nM) Affinity for Ca²⁺

Abstract

Based on studies using high-affinity Ca²⁺ probes (dissociation constant (Kd) = 0.15–0.3 µM), steady-state [Ca²⁺]ⁱⁿ is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca²⁺]ⁱⁿ may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca²⁺. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca²⁺ indicator (Kd ca. 50 µM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca²⁺ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca²⁺. Moreover, pH affects both high and low affinity Ca²⁺ binding site. Since Mag-Fura-2 has been used to study Ca²⁺ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca²⁺ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca²⁺ in specific compartments believed to exhibit high micromolar levels of Ca²⁺.

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…However, an important limitation of this technique is that frequently these dyes, particularly the acetomethoxy ester derivative-type dyes, are prone to compartmentalization in various other intracellular organelles as well (Takahashi et al, 1999). Thus, Hofer and Schulz (1996) have determined that, at least in fibroblasts, only 88% of the loaded low-affinity Ca 2ϩ indicator mag-fura-2 (furaptra) (Martinez-Zaguilan et al, 1998) is restricted to the thapsigargin-sensitive ER. In fact, potential contribution of a confounding signal stemming from its accumulation in mitochondria must be kept in mind (Gurney et al, 2000) (see Section I.D.…”

Imaging approaches to measuring lysosomal calcium

“…The low pH L (4.5e6.5) potentially impacts upon Ca 2þ indicators in two deleterious ways as follows: (1) the chromophore motif may be quenched by acidic pH so that there is insufficient light signal, or (2) the Ca 2þ binding motif is pH-sensitive: the Ca 2þ -binding site of Ca 2þ indicators is often reasonably insensitive to other cations such as Mg 2þ , but it does not exclude H þ which can ably compete with Ca 2þ over these pH ranges. The consequence of H þ competition for the indicator is that it increases the K d for Ca 2þ (i.e., it shifts to a lower Ca 2þ affinity), potentially by orders of magnitude i.e., the K d is pH-dependent (Christensen, Myers, & Swanson, 2002;Martinez-Zaguilan, Parnami, & Martinez, 1998).…”

Scleral Lens Sensor for Ocular Electrolyte Analysis

“…5OACA contains a benzofuran dye (Figure S2, Supporting Information) . 5OACA binds with a 1:1 stoichiometry to Mg 2+ ions ( Figure a) . To test for the selectivity, the 5OACA (25 µmol L −1 ) was mixed with Mg 2+ ion solutions (1–100 mmol L −1 ) (Figure b).…”