Abstract
Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.
Highlights
The most prominent feature of erythrocyte ionic permeability is the selectivity for anions
The dominance of GCl- over the other conductance’s clamps the membrane potential of red blood cells (RBCs) close to the Nernst equilibrium for Cl− (−12 mV), facilitating in this way CO2 transport within the blood owing to fast hydration of this gas within RBCs by the carbonic anhydrase coupled to the fast exchange of produced bicarbonate through the chloride/bicarbonate exchanger AE1 or Band 3, the so-called “chloride shift” (Hoffman and Geibel, 2005)
NS3623 was previously reported as a chloride conductance inhibitor with a higher affinity than that of the hitherto most effective blocker NS1652 (Bennekou et al, 2001)
Summary
The most prominent feature of erythrocyte ionic permeability is the selectivity for anions Hydrophilic anions such as Cl− and HCO−3 cross the membrane about one million times faster than hydrophilic cations of similar size such as Na+ or K+. Studies on chloride channels present in RBCs membrane regained interest when anion channels could potentially be targeted to eradicate malaria (Kirk, 2000). These works done using patch-clamp allowed to revisit the molecular nature of anion channels in RBCs (Egee et al, 2002; Huber et al, 2002). The activation threshold is reached at 150 nM and the maximum activity at around 2 μM (Dunn, 1998); the transmembrane K+ flux is multiplied by a thousand: the massive output of K+ ions hyperpolarizes the membrane, whose potential shifts toward the equilibrium potential for K+ ions (EK) and creates a favorable electrochemical gradient for anion release, leading to fast and massive dehydration
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