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Carboxy-terminal determinants of conductance in inward-rectifier K channels.

Zhang YY, Robertson JL, Gray DA, Palmer LG - J. Gen. Physiol. (2004)

Bottom Line: Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus.The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH.The effects were similar to those predicted for two independent resistors arranged in series.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021, USA.

ABSTRACT
Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.

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Effects of the N240R mutation on a channel with a higher conductance of the selectivity filter. (A) Single-channel traces for C7 chimera and C7 N240R with a holding potential of −80 mV. (B) I–V relationships for C7 and N240R. Single-channel conductances were C7, 58 pS, and C7 N240R, 36 pS.
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fig8: Effects of the N240R mutation on a channel with a higher conductance of the selectivity filter. (A) Single-channel traces for C7 chimera and C7 N240R with a holding potential of −80 mV. (B) I–V relationships for C7 and N240R. Single-channel conductances were C7, 58 pS, and C7 N240R, 36 pS.

Mentions: As a further test for the independence of the conductance properties conferred by the selectivity filter and the COOH terminus, we examined the effect of the N240R mutation on the C7 channels, which were found previously to have a single-channel conductance that is larger than that of ROMK2 itself, presumably due to an increase in conductance through the selectivity filter region (Choe et al., 2000). We reasoned that if the N240R mutation altered the conduction pathway independently of the selectivity filter, then the absolute reduction in single-channel conductance should, if anything, be larger in the C7 background. This prediction was confirmed as shown in Fig. 8. The slope conductance decreased from 58 pS for C7 to 36 pS for C7 N240R. More precisely, if the N240R mutation adds an additional barrier to ion movement that is independent of the selectivity filter, the resistance increase should be similar in both cases. Measuring resistance in the range of −40 to −100 mV, where the I–V curves are reasonably linear in all cases, we calculate that the N240R mutation increases resistance by 8.2 GΩ in the ROMK2 background and by 10.7 GΩ in the C7 background. We considered the two values to be in reasonable agreement. Thus, at least to a first approximation, the effects on conductance of changing the selectivity filter and the COOH terminus are independent.


Carboxy-terminal determinants of conductance in inward-rectifier K channels.

Zhang YY, Robertson JL, Gray DA, Palmer LG - J. Gen. Physiol. (2004)

Effects of the N240R mutation on a channel with a higher conductance of the selectivity filter. (A) Single-channel traces for C7 chimera and C7 N240R with a holding potential of −80 mV. (B) I–V relationships for C7 and N240R. Single-channel conductances were C7, 58 pS, and C7 N240R, 36 pS.
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Related In: Results  -  Collection

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fig8: Effects of the N240R mutation on a channel with a higher conductance of the selectivity filter. (A) Single-channel traces for C7 chimera and C7 N240R with a holding potential of −80 mV. (B) I–V relationships for C7 and N240R. Single-channel conductances were C7, 58 pS, and C7 N240R, 36 pS.
Mentions: As a further test for the independence of the conductance properties conferred by the selectivity filter and the COOH terminus, we examined the effect of the N240R mutation on the C7 channels, which were found previously to have a single-channel conductance that is larger than that of ROMK2 itself, presumably due to an increase in conductance through the selectivity filter region (Choe et al., 2000). We reasoned that if the N240R mutation altered the conduction pathway independently of the selectivity filter, then the absolute reduction in single-channel conductance should, if anything, be larger in the C7 background. This prediction was confirmed as shown in Fig. 8. The slope conductance decreased from 58 pS for C7 to 36 pS for C7 N240R. More precisely, if the N240R mutation adds an additional barrier to ion movement that is independent of the selectivity filter, the resistance increase should be similar in both cases. Measuring resistance in the range of −40 to −100 mV, where the I–V curves are reasonably linear in all cases, we calculate that the N240R mutation increases resistance by 8.2 GΩ in the ROMK2 background and by 10.7 GΩ in the C7 background. We considered the two values to be in reasonable agreement. Thus, at least to a first approximation, the effects on conductance of changing the selectivity filter and the COOH terminus are independent.

Bottom Line: Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus.The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH.The effects were similar to those predicted for two independent resistors arranged in series.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021, USA.

ABSTRACT
Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at V(m) = -100 mV from 34 to 21 pS. The slope conductance measured between -60 and -140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba(2+), suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl(+):K(+), accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K(+) transport.

Show MeSH
Related in: MedlinePlus