Physiology and Pathophysiology of the Erythrocyte Gardos Channel in Hematological Diseases

1. Abstract

The erythrocyte intermediate conductance Ca-activated K channel (IK1), first described by Gardos, has been shown to play an important role in the pathological dehydration of human and mouse sickle erythrocytes. Studies by various groups have demonstrated K loss mediated by the activation of this pathway when sickle erythrocytes are subjected to deoxygenation. Transient, localized increases in cytoplasmic Ca induced by sickling are a possible mechanism for this activation. In addition, it has been shown that the Gardos channel of sickle and normal erythrocytes can be activated in oxygenated conditions by PGE₂, endothelin-1, and chemokines. Since many of these biological mediators are increased systemically and locally in sickle cell disease, it is possible sickle cells may also dehydrate in the absence of deoxygenation. Studies on the Gardos channel of sickle erythrocytes have led to testing of specific inhibitors in vivo: the imidazole antimy-cotic clotrimazole (CLT) showed promising cellular effects in vivo which led to the development of a novel compound (ICA-17043) currently in phase II clinical trials.1 In vivo studies with clotrimazole in normal human subjects and control mice showed no significant changes in erythrocyte volume and K content, supporting the idea that the Gardos channel does not play a role in regulating ion content of normal cells. However, the Gardos channel seems to play a role in controlling cell volume in other hematological conditions: Halperin et al.2 have shown that complement-induced hemolysis is modulated and reduced by K loss via the Gardos pathway. A role of the Gardos channel in hereditary spherocytosis (HS) has been advocated for a long time but with little experimental evidence. We have studied the ion transport properties and the effects of in vivo Gardos channel blockade in a mouse model with complete deficiency of all 4.1 proteins isoforms.3 4.1-/- erythrocytes exhibit cell dehydration, with reduced cell K content and markedly increased Na content and permeability to Na, mostly mediated by Na/H exchange. The Na/H exchange of 4.1-/- cells is markedly activated by exposure to hypertonic conditions and exhibits an abnormal dependence on osmolarity and internal pH. In 4.1 -/- erythrocytes, the V_max of the Gardos channel is significantly higher than in controls (from 9.75 ± 1.06 vs. 6.08 ± mmol/L cell x min, p<0.04). In addition, 4.1 -/- erythrocytes showed a significantly lower affinity constant for internal Ca²⁺ (from 1.47 to 1.01 μM, p<0.03). When 4.1 -/- mice were treated with oral CLT, a Gardos channel blocker, worsening of anemia, increased mortality and increased cell dehydration/ fragmentation were noted. Essentially similar but less severe changes in red cell features were obtained in vivo with a CLT analog devoid of the imidazole moiety in 4.1 -/- mice, and with CLT in 4.2-/-, and band 3 -/+ mice. The present data indicate that K and water loss via the Gardos channel may play a crucial role in compensating for the reduced surface membrane area of murine HS erythrocytes and protecting erythrocytes from lysis.