Mechanism of Ba²⁺ block of a mouse inwardly rectifying K⁺ channel: differential contribution by two discrete residues

Abstract

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...

Three Mechanisms Underlie KCNQ2/3 Heteromeric Potassium M-Channel Potentiation

“…Mutational analysis in KCNQ2, K ir 2.1 and Shaker is consistent with the involvement of T276/A315 in an intracellular ionic block (Yellen et al, 1991;Zhou et al, 1996;Alagem et al, 2001;Slesinger, 2001;Prole and Marrion, 2004). Indeed, the corresponding T623 is critical for anti-arrhythmic drug blockage of human ether-à-go-go related gene channels (Mitcheson et al, 2000), and in KCNQ1 there are important determinants for benzodiazepine channel block in the neighborhood of this residue (Seebohm et al, 2001).…”

Targeting potassium channels to treat cerebellar ataxia

“…Chlorzoxazone does not likely act through SK channels in the dendrites, since SKA‐31 had no effect on dendritic excitability. When tested in the presence of barium (50 μ mol/L), which at this dose selectively blocks subthreshold‐activated inwardly rectifying potassium (K ir ) channels,2, 50, 51, 52, 53, 54 the effect of chlorzoxazone on reducing dendritic excitability was prevented (Fig 5G) (input resistance ATXN1[82Q] + TTX + Barium 47.8 ± 6.8, ATXN1[82Q] + TTX + Barium + Chlorzoxazone 57.1 ± 3.8, P = 0.173). This suggests that chlorzoxazone likely activates K ir channels in the dendrites of ATXN1[82Q] Purkinje neurons to reduce dendritic hyperexcitability.…”

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

“…6). Our results show only small differences from those of Alagem et al (2001). Using oocyte expression and working with higher [K ＋ ]o, they found a Kd(0) of 320 ± 18μM…”

“…Mutants of T141 and S165 affect the deeper binding site, whereas the residues R148 or E125, at the outer mouth of the channel (Navaratnam et al, 1995;Doring et al, 1998;Krapivinsky et al, 1998;Topert et al, 1998), are likely to form the shallow site. Alagem et al (2001) also dissected two sites, one at the outer mouth, at E125 (Alagem et al, 2001). Jiang & MacKinnon (2000) (see also Neyton & Miller, 1988a, b) argued that K ＋ occupancy of the central cavity of KcsA would lock Ba 2＋ into its blocking site.…”

“…The mutant channel Kir 1.1 (V121T) exhibited a ten-fold increase in Ba 2＋ affinity. In Kir2.1, Alagem et al (2001) implicated Thr141 as a determinant of Ba 2＋ blockage. Involvement of the pore helix was demonstrated in Kir3.0 by Lancaster et al (2000), who showed that mutation of residues of Kir3.1/3.4 heteromers (F137S and E139Q in Kir3.1 and E145Q in Kir3.4) produced substantial decreases in Ba 2＋ sensitivity.…”