Fluorescent imaging of Cl ^- in Amphiuma red blood cells: How the nuclear exclusion of Cl ^- affects the plasma membrane potential

Abstract

In this work, we test the idea that most, if not all, cellular Cl ؊ of Amphiuma red blood cells is contained in the cytoplasm. If true, this could resolve the difference between the measured plasma membrane potential (E m) and that expected from the Donnan equilibrium distribution of Cl ؊ . We studied the changes in the fluorescence intensity of the Cl ؊ -sensitive dye, MQAE, entrapped in red cells that occurred when intracellular Cl ؊ was exchanged with NO 3 ؊ . We could thus monitor the distribution of Cl ؊ between the nuclear and cytoplasmic compartments. We found that essentially all of the cell's Cl ؊ resides in the cytoplasm. Knowing the volume of the cell occupied by the nucleus, we could accordingly correct the measured values of cell Cl ؊ . This resulted in establishing a concordance between the measured values of E m and those calculated from the corrected values of the Cl ؊ ratio, thus explaining the discrepancy. The exclusion of Cl ؊ from the nucleus may result from its unusually high content of ''excess'' DNA that imposes an imbalance of net negative charge.MQAE ͉ nuclear͞cytoplasmic ratios T he equilibrium distribution of permeable ions such as Cl Ϫ across the plasma membrane of red blood cells should be determined by the membrane potential (E m ) that is set by the relative permeability to Na ϩ and K ϩ and their concentration gradients. This paper addresses the E m of Amphiuma red blood cells and the reason why the measured E m appears to differ significantly from that expected from the equilibrium distribution of Cl Ϫ . The idea that the E m of red blood cells in general and of human red cells in particular could be described by a Donnan potential is based on analyses (1-4) of the passive equilibrium distribution of permeable ions between the cytoplasm (Cl i Ϫ ) and the external medium (Cl o ). The E m of human red cells, measured indirectly with the use of fluorescent dyes (5), was found to vary quantitatively with the Donnan ratio of Cl i ͞Cl o (r Cl ). This was so when r Cl was varied either by changes in external pH (pH o ) (5-7) or by altering the concentration of Cl o by substitution with an impermeable anion (5). Validation that the fluorescence technique provided an accurate assessment of E m in human red cells stemmed from the concordance of E m determined directly in Amphiuma red blood cells by impalement with microelectrodes (8, 9, †) with those determined by the use of fluorescent dyes (5). These results established the basis for expecting measurements of E m in Amphiuma red blood cells to conform to a Donnan potential. However, analyses showed that, although the E m of Amphiuma responded to changes in Cl i produced by changes in pH o , the calculated values of E m based on measured values of Cl i and Cl o were significantly higher than the observed E m values. E m was calculated by use of the Nernst equation, E m ϭ 58 log Cl i ͞Cl o , where 58 is the value of RT͞F at 20°C., with R being the gas constant, T the temperature, and F, the Faraday constant. It turns out that the problem li...

My Passion and Passages with Red Blood Cells

“…We saw that we could resolve the discrepancy by assuming that all the cellular Cl − was contained in the cytoplasm and that none was in the nucleus. Some 35 years later, John Geibel and I showed that the nucleus does indeed exclude Cl − , presumably because of its high content of negatively charged noncoding DNA (63). These results provided concordance between the measured E m s and those calculated from the now-modified Donnan ratios (63).…”

“…Some 35 years later, John Geibel and I showed that the nucleus does indeed exclude Cl − , presumably because of its high content of negatively charged noncoding DNA (63). These results provided concordance between the measured E m s and those calculated from the now-modified Donnan ratios (63). While carrying out the aforementioned measurements, Ulrik and I encountered a perplexing problem: We were unable to observe positive changes in E m s upon the substitution of external Cl − with an impermeant anion, p-amino-hippurate.…”

Functional KV10.1 Channels Localize to the Inner Nuclear Membrane

“…If it would not be permeable to ions, the ionic composition should be different between the nucleoplasm and cytoplasm, and there could be an electrical potential across the nuclear envelope. However, there are few reports on the ion composition of the nucleoplasm [25] , [26] and, in most cases, the intranuclear potential is only slightly negative compared to the cytoplasm [20] but is still dependent on the cytoplasmic K + concentration [27] . On the other hand, there are compelling electrophysiological studies where it has been proposed that the NPC can close in a voltage-dependent manner in the absence of cargo, and that ion flow is prevented when the pore is occluded by cargo [28] .…”

Inward Tension of Talin and Integrin-related Osmotic Pressure are involved Synergetically in the Invasion and Metastasis of Non-small Cell Lung Cancer

“…2A). Furthermore, (6-methoxyquinolinio)acetic acid ethyl ester bromide (MQAE) was used to investigate the changes in intracellular Cl - concentration 31 , 32 . The data indicate that the increased Cl - content is closely associated with the upregulation of protein nanoparticles (Fig.…”