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  • More recently NMR techniques have been used to monitor


    More recently, NMR techniques have been used to monitor K+ () fluxes in isolated rat hearts. These reveal an increased rate of passive efflux of from Langendorff-perfused hearts at 20°C compared to 36°C or 10°C (Gruwel, Kuzio, Xiang, Deslauriers, & Kupriyanov, 1998). Similar experiments assessed the activity and inhibition of ATP-sensitive K+ channels under physiological and pathological conditions (Kupriyanov et al., 1998, Kupriyanov et al., 1999) and probed the mitochondrial K+ pool without isolation from the RGDfK (Gruwel, Kuzio, Deslauriers, & Kupriyanov, 1999). Analogous NMR techniques may provide information in the investigation of the contribution of Ca2+-activated K+ channels to relevant organs or tissues. NMR of erythrocyte suspensions is well established, with several studies investigating the interaction of Cl− with the band 3 protein (Falke et al., 1984, Price et al., 1991, Liu et al., 1996). Net transport of chloride ions across the plasma membrane of these cells, such as that which occurs as a consequence of Gárdos channel activation, can be monitored by NMR. Like monovalent cations, the NMR signal from erythrocytes consists of coincident resonances for intra- and extra-cellular nuclei. To add to this, the time constant for transverse relaxation of chloride inside the cell is much shorter than outside, leading to a very broad intracellular signal. This potential disadvantage may be circumvented by appropriate adjustment of the NMR spectrum such that it is representative only of the extracellular Cl− (Brauer, Spread, Reithmeier, & Sykes, 1985), or by employing a Co2+ shift reagent (Shachar-Hill & Shulman, 1992) to separate the intra- from the extra-cellular signal. With the chloride permeability of erythrocytes now identified as a therapeutic target in sickle cell anaemia (Bennekou et al., 2001; Bennekou, Pedersen, Moller, & Christophersen, 2000; Joiner et al., 2001a), NMR may provide a convenient, non-invasive means of monitoring Gárdos channel activity.
    Conclusions From its initial description to the present day, much has been learnt with regard to the role of the Gárdos channel in both physiological and pathophysiological states of the human erythrocyte. The activity of this K+ channel is influenced from both sides of the membrane by myriad biochemical and biophysical stimuli, both endogenous and exogenous. While the details of the interactions of compounds that cooperate to modulate the channel’s behaviour continue to be investigated, several conclusions can be drawn from the available evidence:
    Introduction Dehydration of sickle red blood cells (SSRBCs), resulting from an intracellular depletion of potassium (K+), promotes hemoglobin S (HbS) polymerization and contributes to both the vaso-occlusive and hemolytic aspects of sickle cell anemia (SCA) [1], [2]. Different pathways are involved in this process, mainly the deoxygenation-induced cation pathway (Psickle) [3], the KCl cotransport (KCC) [4], and the Ca-dependant K+ channel (Gardos channel) [5], [6], [7]. The Gardos channel appears as a major contributor to the dehydration of erythrocytes of SCA patients and has long been recognized as a potential therapeutic target for this disease. Several triarylamides have been identified as potent inhibitors of the channel and active in mouse model of SCD. Among them, Senicapoc (also called ICA-17043) may be beneficial for the treatment of hemolysis-associated complications of the disease [8], [9] but failed to reduce the frequency of vaso-occlusive crises. A better understanding of the Gardos channel regulation could lead to the development of more efficient therapeutic strategy. Recent research has focused on the various cellular conditions and compounds that influence the Gardos effect in normal and sickle cells [10], [11]. Evidence based on kinetic analysis of the Gardos channel activity has indicated that the cytokines RANTES and IL-10, as well as the proinflammatory molecules such as ET-1 and PAF, modify the affinity constant for intracellular Ca2+ and increase the apparent maximal velocity (Vmax) of the channel transport in normal erythrocytes [10], [12]. The effect of RANTES, a CC chemokine, was not observed in normal erythrocytes lacking the Duffy antigen (Duffy-negative erythrocytes). Moreover, the authors showed that RANTES enhanced the formation of dense cells in Duffy-positive but not in Duffy-negative SSRBCs. The erythrocyte Duffy antigen (Fy), nowadays known as the Duffy antigen receptor for chemokines (DARC), binds chemokines of both the CC and CXC families [13]. The Duffy-negative phenotype Fy (a−b−), which is rare among Caucasians, is present in most West African (>95%) and in approximately 68% of African American individuals [13].