Neutralization of aspartate residues in the murine inwardly rectifying K+channel IRK1 affects the substate behaviour in Mg2+block

Autor: Keiko Oishi, Koh Shingu, Hideo Ohyama, Hiroko Matsuda, Koichiro Omori
Rok vydání: 1998
Předmět:
Zdroj: The Journal of Physiology. 510:675-683
ISSN: 0022-3751
DOI: 10.1111/j.1469-7793.1998.675bj.x
Popis: To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single-channel currents through inwardly rectifying K+ (IRK1) channels were studied. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude seen in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. Both mutations reduced the Mg2+ sensitivity of the channel at 2 μm Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 μm) and voltages (+70 mV). Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single-barrelled channel. The phenomenon of inward rectification, whereby the K+ conductance increases under hyperpolarization and decreases under repolarization, has been demonstrated in a variety of cell types (Katz, 1949; Hall et al. 1963; Kandel & Tauc, 1966; Hagiwara & Takahashi, 1974). This behaviour plays an important role in permitting long depolarizing responses. Studies on native cardiac channels and cloned inwardly rectifying K+ (IRK1) channels (Kubo et al. 1993) indicate that inward rectification mainly results from a voltage-dependent block of the channel pore by intracellular Mg2+ (Matsuda et al. 1987; Vandenberg, 1987; Matsuda, 1988) and polyamines (Ficker et al. 1994; Lopatin et al. 1994; Fakler et al. 1995). The key feature of a Mg2+ block of the cardiac inwardly rectifying K+ channel is that in the presence of intracellular Mg2+ at micromolar concentrations the outward single-channel current fluctuates between four equally spaced conductance levels, including zero current (Matsuda, 1988, 1991a). Three distinct blocked states are also seen during blockade by intracellular Ca2+ (Matsuda & Cruz, 1993) and extracellular Cs+ or Rb+ (Matsuda et al. 1989). The distribution of the current levels agreed reasonably well with the binomial theorem at different probabilities for the blocked state. The substate behaviour can be most simply explained by assuming that the inwardly rectifying K+ channel consists of three identical conducting units that usually function co-operatively to form a single channel and that blocking ions enter and plug each conducting unit independently. Another mechanism that could induce sublevels involves different binding patterns of the blocking ions to sites on the channel protein, leading to different conductive states in a single-barrelled channel. We confirmed that intracellular Mg2+ induces sublevels in channels expressed by transfecting COS-1 cells with the IRK1 gene, as in native channels (Omori et al. 1997). In the present study, we report that a negatively charged residue at position 172 in the second hydrophobic segment of the IRK1 channel is essential for the substate behaviour, favouring the latter mechanism proposed above. A model to explain the substate behaviour in a single-barrelled channel is proposed.
Databáze: OpenAIRE
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