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Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels.

Yeh SH, Chang HK, Shieh RC - J. Gen. Physiol. (2005)

Bottom Line: These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel.In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel.We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.

ABSTRACT
Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

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MTSET remains accessible to T141C when spermine inhibits K+ efflux in the T141C/D172N mutant. Current traces in the T141C/D172N mutant exposed to the control (A) and 100 μM spermine (B) solution. Current was recorded at −140 mV from a holding potential of +40 mV before (solid line) and after (dotted line) MTSET treatment (1 mM). The number of seconds on the bottom of each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the right panels. The horizontal lines indicate the zero current levels. (C) In the presence of 100 μM spermine, currents were monitored for 50 s before MTSET treatment. MTSET was applied for 100 s as the patch was held at +40 mV without pulsing. Thereafter, MTSET was washed out and currents were recorded with brief hyperpolarizing pulse to −140 mV. (D) Time courses of MTSET reaction in T141C mutants in control and spermine as indicated.
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fig3: MTSET remains accessible to T141C when spermine inhibits K+ efflux in the T141C/D172N mutant. Current traces in the T141C/D172N mutant exposed to the control (A) and 100 μM spermine (B) solution. Current was recorded at −140 mV from a holding potential of +40 mV before (solid line) and after (dotted line) MTSET treatment (1 mM). The number of seconds on the bottom of each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the right panels. The horizontal lines indicate the zero current levels. (C) In the presence of 100 μM spermine, currents were monitored for 50 s before MTSET treatment. MTSET was applied for 100 s as the patch was held at +40 mV without pulsing. Thereafter, MTSET was washed out and currents were recorded with brief hyperpolarizing pulse to −140 mV. (D) Time courses of MTSET reaction in T141C mutants in control and spermine as indicated.

Mentions: Our previous study suggests that spermine, interacting with E224 and E299, inhibits K+ efflux by blocking at a position between 141 and 164 or by other mechanisms (Chang et al., 2003). To test the former possibility we examined whether the inhibition of T141C/D172N mutants by spermine can prevent MTSET from accessing the substituted cysteines at site 141. To avoid trapping the reagent in the pore following brief channel openings, membrane patches were held at +40 mV and stepped to −140 mV for a short period (5 ms) at 0.2 Hz to monitor channel activities. Using such a protocol, the open probability in the presence of spermine is 0.001, low enough to avoid trapping the reagent (Phillips et al., 2003). In the control, outward currents were observed and MTSET progressively inhibited the T141C/D172N mutant (Fig. 3 A). Spermine inhibited the outward current recorded at the holding potential (Fig. 3 B), presumably due to channel inhibition effected by spermine binding to E224 and E299 (Yang et al., 1995; Kubo and Murata, 2001). However, MTSET remained capable of irreversibly inhibiting the T141C/D172N mutant, indicating that spermine does not block the pore internal to the site 141.


Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels.

Yeh SH, Chang HK, Shieh RC - J. Gen. Physiol. (2005)

MTSET remains accessible to T141C when spermine inhibits K+ efflux in the T141C/D172N mutant. Current traces in the T141C/D172N mutant exposed to the control (A) and 100 μM spermine (B) solution. Current was recorded at −140 mV from a holding potential of +40 mV before (solid line) and after (dotted line) MTSET treatment (1 mM). The number of seconds on the bottom of each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the right panels. The horizontal lines indicate the zero current levels. (C) In the presence of 100 μM spermine, currents were monitored for 50 s before MTSET treatment. MTSET was applied for 100 s as the patch was held at +40 mV without pulsing. Thereafter, MTSET was washed out and currents were recorded with brief hyperpolarizing pulse to −140 mV. (D) Time courses of MTSET reaction in T141C mutants in control and spermine as indicated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266598&req=5

fig3: MTSET remains accessible to T141C when spermine inhibits K+ efflux in the T141C/D172N mutant. Current traces in the T141C/D172N mutant exposed to the control (A) and 100 μM spermine (B) solution. Current was recorded at −140 mV from a holding potential of +40 mV before (solid line) and after (dotted line) MTSET treatment (1 mM). The number of seconds on the bottom of each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the right panels. The horizontal lines indicate the zero current levels. (C) In the presence of 100 μM spermine, currents were monitored for 50 s before MTSET treatment. MTSET was applied for 100 s as the patch was held at +40 mV without pulsing. Thereafter, MTSET was washed out and currents were recorded with brief hyperpolarizing pulse to −140 mV. (D) Time courses of MTSET reaction in T141C mutants in control and spermine as indicated.
Mentions: Our previous study suggests that spermine, interacting with E224 and E299, inhibits K+ efflux by blocking at a position between 141 and 164 or by other mechanisms (Chang et al., 2003). To test the former possibility we examined whether the inhibition of T141C/D172N mutants by spermine can prevent MTSET from accessing the substituted cysteines at site 141. To avoid trapping the reagent in the pore following brief channel openings, membrane patches were held at +40 mV and stepped to −140 mV for a short period (5 ms) at 0.2 Hz to monitor channel activities. Using such a protocol, the open probability in the presence of spermine is 0.001, low enough to avoid trapping the reagent (Phillips et al., 2003). In the control, outward currents were observed and MTSET progressively inhibited the T141C/D172N mutant (Fig. 3 A). Spermine inhibited the outward current recorded at the holding potential (Fig. 3 B), presumably due to channel inhibition effected by spermine binding to E224 and E299 (Yang et al., 1995; Kubo and Murata, 2001). However, MTSET remained capable of irreversibly inhibiting the T141C/D172N mutant, indicating that spermine does not block the pore internal to the site 141.

Bottom Line: These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel.In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel.We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.

ABSTRACT
Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

Show MeSH
Related in: MedlinePlus