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Abstract
BackgroundThe mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.Methodology/Principal FindingsThe cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.Conclusions/SignificanceThe stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.
Highlights
The cell-attached configuration of the patch-clamp technique respects the integrity of the intracellular milieu
NaCl, pCa = 3) in the pipette and bathing solutions (56 patches), RBC membranes seldom displayed channel activity when recordings were performed more than 10–15 min after seal formation (5 out of 56), at the spontaneous membrane potential, i.e. at pipette potential of zero mV (-Vp = 0 mV), whatever the degree of membrane deformation induced by underpressure
Response Gardos channel activation required the presence of calcium in the medium but not in the patch pipette suggesting that local deformation somehow activated Ca2+ permeability pathways, pathway with a finite Ca2+ conductance (PCa), distant to the domed area within the patch pipette, leading to increased [Ca2+]i
Summary
The cell-attached configuration of the patch-clamp technique respects the integrity of the intracellular milieu. It reflects best the physiological condition of the currents recorded across the membrane patch trapped within the tip of the microelectrode. Once the seal is established, the membrane deformation induced by the glass pipette, regardless of the intensity of underpressure, is not under experimental control, and may vary from one cell to another. In our previous studies in intact red cells we seldom observed spontaneous channel activity in cell attached patches when the cells were bathed in physiological saline solution. Intrigued by the systematic link between the negative pressure pulse and the transient current response, we explored some of the medium requirements in preliminary experiments. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents
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