Inwardly rectifying potassium (Kir) channels are involved in many physiological processes, such as setting the excitability state of nerve and muscle, potassium secretion and hormone release. They act by allowing the flux of potassium ions near the potassium equilibrium potential, thus keeping the resting membrane potential hyperpolarized. The inward rectification is attributed to a voltage-dependent block of the channel pore by intracellular magnesium and polyamines (Fakler et al. 1995;Lopatin et al. 1995).Inorganic cations have been widely used to probe the permeation and gating mechanisms of potassium channels (Hille, 1992 (Standen & Stanfield, 1978, 1980 Ohmori, 1978;Biermans et al. 1987;Harvey & Ten Eick, 1989; Shioya et al. 1993; Reuveny et al. 1996; Sabirov et al. 1997a; Shieh et al. 1998;Doring et al. 1998;Dart et al. 1998). The interaction of divalent cations with Kir channels is thought to occur via two distinct binding sites; a shallow site that barely senses the membrane electric field, and a deeper one located approximately half-way within the membrane electrical field. Channel block by Mg 2+ and Ca 2+ ions was found to occur through the shallow site, whereas the block by Ba 2+ and Sr 2+ ions is mediated through the deeper one (Standen & Stanfield, 1978; Shioya et al. 1993; Reuveny et al. 1996; Sabirov et al. 1997b; Shieh et al. 1998). For all divalent cations, a single ion suffices to block the channel. In most cases of deepsite blockers, the block follows first-order kinetics, taking several seconds to reach a steady-state (Standen & Stanfield, 1978; Shieh et al. 1998). An exception is the G-protein-coupled inwardly rectifying potassium channel family, where part of the Ba 2+ block reaches steady state in an unmeasurably short time (Carmeliet & Mubagwa, 1986).Since the recent cloning of many Kir channels, some progress has been made in understanding the molecular mechanisms involved in the channel block by divalent cations. Sabirov et al. (1997b) showed that a highly conserved arginine residue at position 148 in Kir2.1/IRK1 forms a barrier for external cations. Mutating R148 to histidine allowed Mg 2+ and Ca 2+(shallow blockers) to bind more deeply within the electric field. Block by Ba 2+ and Cs + became more rapid, while the affinity and voltage dependence of the block remained unchanged. Additional information related to the role of this conserved arginine in channel block came from a unique member of the Kir channel family, Kir7.1. This channel has a methionine at the position corresponding to that of the conserved arginine. This methionine was 1. The block of the IRK1/Kir2.1 inwardly rectifying K + channel by a Ba 2+ ion is highly voltage dependent, where the ion binds approximately half-way within the membrane electrical field. The mechanism by which two distinct mutations, E125N and T141A, affect Ba 2+ block of Kir2.1 was investigated using heterologous expression in Xenopus oocytes.2. Analysis of the blocking kinetics showed that E125 and T141 affect the entry and binding of Ba 2+ t...