A critical role for altered red cell cation permeability in pathogenesis of sickle cell disease and other haemolytic anaemias

The aetiology of sickle cell disease is well known, but pathogenesis is complicated and details remain uncertain. A thorough understanding may suggest novel ways for designing more effective therapies. One area of importance, covered here in Nader et al., is the altered cation permeability of sickle cells and how the co‐ordinated operation of a number of membrane transport proteins contributes to disease progression, all driven by the initial event of HbS polymerisation. There are echoes here of the cation leaks of hereditary stomatocytosis. Nader et al. propose a central role for PIEZO1, a novel mechanosensitive channel found in red cells, which may be aberrantly activated in sickle cells following HbS polymerisation and which may have potential as a novel target for future chemotherapies.

A key component of red cell homeostasis is a membrane, which usually presents with a very modest permeability to cations. Any change may potentially result in unbalanced ionic fluxes, which can decrease or increase volume, compromise longevity and cause many other problems. Notwithstanding, there are a number of important cation transport systems present in the membrane and their integrated function is pivotal to red cell pathophysiology, including that in sickle cell disease (SCD). Some of these have only recently been identified, whilst others are doubtless yet to be discovered. The rich complexity of their interactions is the subject of much recent research. 1 One such transport system is Piezo1, a giant protein of some 300 kDa and over 30 membrane-spanning domains. Piezo1 is best known as a fairly non-selective mechanosensitive cation channel. 2 It has several important roles in the circulatory system, including vascular development, as well as in red cell behaviour.
In their paper the authors 3 consider the role of Piezo1, integrated with that of other red cell cation channels, in SCD pathogenesis. Its function in sickle cells is explored using pharmacological activation and inhibition of Piezo1 (with Yoda1 and tarantula venom, GsMTx4, respectively), measurements of deformability, electrophysiology, intracellular calcium fluorophores/chelation, indices of stickiness, including phosphatidylserine (PS) exposure and the role of other adhesion molecules, and the behaviour of sickle cells from a patient with a gain-of-function Piezo1 mutation. The authors interpret their findings as showing that Piezo1 activation following red cell distortion by HbS polymers (or by Yoda1) mediates Ca 2+ entry and a number of important sequelae. These include the activation of the Ca 2+ -activated K + channel or Gárdos channel, subsequent loss of K + , with Cl − following through separate anion channels, consequent red cell shrinkage and hyperpolarisation, with altered activity of voltage-sensitive cation channels, with further permeability changes, possibly including enhanced Ca 2+ entry and increased scrambling of PS. Their figure 4 presents a schematic summary of these events.

C O M M E N T A R Y A critical role for altered red cell cation permeability in pathogenesis of sickle cell disease and other haemolytic anaemias
The aetiology of sickle cell disease is well known, but pathogenesis is complicated and details remain uncertain. A thorough understanding may suggest novel ways for designing more effective therapies. One area of importance, covered here in Nader First is work on the so-called 'P sickle ' pathway of sickle cells. Our understanding of this enigmatic permeability dates from the seminal work of Tosteson in the 1950s. It is considered to be a non-selective cation conductance activated upon deoxygenation, HbS polymerisation and shape change. 4 P sickle activation is associated with Ca 2+ gain, Mg 2+ loss, KCl loss, shrinkage and PS scrambling, amongst other deleterious sequelae. Like Piezo1, P sickle also appears to be inhibited by GsMTx4 and other possible mechanosensitive channel blockers (e.g. Refs [5,6]). The selection pressure for the sickle mutation is malaria resistance, and interestingly, a gain-of-function Piezo1 mutation (E756del) is widely present in African populations where it is associated with both red cell dehydration and malaria protection 7 -and, in Nader et al., with increased sickling tendency and PS exposure.
Second, as the sickle mutation does not reside in a transport gene, any gene encoding for the P sickle conductance must necessarily be present in non-SCD individuals. In this context, it is notable that normal red cells also appear to open a Ca 2+ permeability on mechanical distortion, as seen for P sickle in deoxygenated sickle cells and for Piezo1 in red cells mechanically distorted by means other than by HbS polymers. In normal red cells, Johnson and others in the 1990s showed how shear stress results in Ca 2+ entry whilst, more recently, in important electrophysiological studies, Thomas and co-workers at Roscoff showed that in single cells, mechanical distortion led to a transient Ca 2+ conductance, like that seen for Piezo1, and which also has a proposed link to Gárdos channel activation, coupled with that of separate anion channels. 8 It is not surprising that Piezo1 has been postulated as an obvious candidate for this pathway. 2,9 Finally, there is also a link with the hereditary stomatocytosis (HSt). A gain-of-function mutation in Piezo1 is implicated in perhaps half of the patients with this form of haemolytic anaemia. 9,10 In HSt, increased entry of Ca 2+ through the mutated Piezo1 is also associated with red cell dehydration via Gárdos channel activation. Other types of HSt are linked to mutations in different ion channels (including that of the Gárdos channel) or even in transporters (like AE1/band 3 or GLUT1), which are normally very distinct transport entities to channels. In HSts, the cation leak is a transport mutation, whilst, of course, in SCD, the mutation is in a haemoglobin gene, HBB. But the principle of unregulated changes in membrane permeability resulting in pathology is common to both.
There are caveats and important areas where our understanding is considerably lacking. These include that of drug specificity. For example, as discussed in Nader et al., Yoda1, in particular, can elicit PS exposure in the complete absence of Ca 2+ . 11 In addition, E756del did not appear to correlate with P sickle activity. 12 And GsMTx4 whilst inhibiting deoxygenationinduced currents in single red cells does not appear to block P sickle in flux experiments and is recognised to be a particularly fickle inhibitor. Possibly Piezo1 behaves differently in single-channel experiments and mixed cell populations.
The work of Nader et al. concerns at least four channels (Piezo1, the Gárdos channel, an anion channel and possibly a voltage-activated non-selective cation channel) or five, if the molecular identity of P sickle is found to be a separate protein to these others. Proteomic analysis indicates that there are many more channels in red cells (e.g. Ref. [13]), as well as hundreds of transporters, and that the complement of these changes during erythropoiesis. Many are regulated by phosphoresidues of serine-threonine and tyrosine residues, which are also subject to change. The complexity of red cell behaviour is perhaps best illustrated with our developing understanding of how AE1/band 3 co-ordinates red cell function and how it is altered in disease states like SCD and malaria invasion (e.g. Ref. [14]).
Whilst the story remains tantalisingly incomplete, the findings of Nader et al. published here emphasise the importance of the co-ordinated function of permeability pathways for red cell pathophysiology. They also highlight the significance of red cell cation transport, especially through channels, as an area of importance to practical clinical haematology, as well as to more arcane areas of red cell research.