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  • Introduction br Channel activity Since initial reports of th

    2022-09-30

    Introduction
    Channel activity Since initial reports of the Ca2+-activated K+ permeability of erythrocytes (see Gardos, 1958), several ‘criteria’ have been established to certify K+ fluxes as being due to the Gárdos channel. For example, the requirement for Ca2+ at the intracellular face of the channel ((Ca2+)i); a single channel conductance of ∼20pS (at 0mV); high selectivity of K+ over Na+; evidence of open channel inward rectification; gating independence of membrane potential; inhibition either by specific drugs such as clotrimazole and charybdotoxin; or inhibition on pre-incubation of Candesartan in the absence of external potassium ((K+)o). These identification criteria can be, and have been, subjected to modulation by various physiological conditions and experimental designs.
    Cloning The first intermediate-conductance Ca2+-activated K+ channel (IK) to be cloned was from human pancreas (designated hIK1) (Ishii et al., 1997). Structurally similar to the SK (small conductance Ca2+-activated K+ channels) subfamily (42–44% sequence identity), the gene product was distinguished as an IK, based on its pharmacological and electrophysiological properties in Xenopus oocytes. Northern blot analysis showed expression of hIK1 in several peripheral body tissues, predominantly smooth muscle. The cDNA sequence predicts a six-transmembrane domain protein with a characteristic pore-region containing the canonical K+ selectivity filter sequence, GYG. The gene encoding the hIK1 protein (called KCNN4) maps to q13.2 on chromosome 19 (Ghanshani et al., 1998). Patch clamp recordings of overexpressed hIK1 in Xenopus oocytes has confirmed the dependence on Ca2+ of the K+ current (K0.5∼0.32μM). The current–voltage relationship (I–V) shows a single channel conductance of 42pS. This is higher than previously reported values (Grygorczyk et al., 1984, Leinders et al., 1992), partly due to the region of the I–V curve that is sampled for calculation of single channel conductance, and possibly also due to heterogeneous distribution of hIK1 unitary conductances between (and within) various tissue types. The protein also exhibits a pharmacological response that is consistent with the Gárdos channel being blocked by clotrimazole and charybdotoxin (IC50∼24.8nM and 2.5nM, respectively) but resistant to iberiotoxin and apamin that are blockers of BK (large conductance Ca2+-activated K+ channels) and SK channels, respectively. Thus firm evidence exists that this gene translates to the Gárdos channel protein in erythrocytes, and advances the prospect of gaining knowledge on its role in other cells including the erythrocyte. The mouse homologue of the gene, mIK1, was cloned and overexpressed in a similar fashion (Vandorpe et al., 1998). The protein shows comparable pharmacology and electrophysiology to hIK1. A regulatory volume decrease is observed in oocytes expressing mIK1 following mild hypotonic swelling of the cells that is associated with a gradual accumulation of (Ca2+)i, and diminishes in the presence of clotrimazole. The same phenomenon is not explicitly observed in human erythrocytes, possibly because they lack the necessary cellular machinery. mIK1 mRNA was shown to be as abundant in the spleen as in murine erythroleukemia (MEL) cells. Treatment of liquid cultures of human CD34−/CD38+ stem cells with clotrimazole inhibits proliferation and retards differentiation of the cells to maturity. Thus, it is apparent that the Gárdos channel plays a role in early erythroid development.
    Inhibitors Since the discovery of the Gárdos effect, there has been an ongoing search for potent and specific blockers of its transport action, especially since it is identified as a major contributor to the dehydration of erythrocytes of patients with sickle cell anaemia. Specific pharmacological characterisation of it is also important in distinguishing between the three different types of Ca2+-activated K+ channels (large, medium and small conductance) in other cells. The drive for increased potency and specificity of Gárdos channel blockers has produced a host of pharmacological ‘tools’ that can be used to study it and other channels. Different blockers have different modes of action, and comprehensive understanding of the modes of action is critical in deciding which inhibitor to use in a particular situation, be it basic experimental or clinical.