Limits...
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.

Show MeSH
Effect of charge at position 240 on single-channel conductance. Conductances measured between −40 and −140 mV were ROMK2, 43 pS; C13, 31 pS; N240R, 31 pS; N240K, 37pS; and N240E, 45pS.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2234023&req=5

fig9: Effect of charge at position 240 on single-channel conductance. Conductances measured between −40 and −140 mV were ROMK2, 43 pS; C13, 31 pS; N240R, 31 pS; N240K, 37pS; and N240E, 45pS.

Mentions: To examine the mechanism of the effect of the N240R substitution on conductance, we tested for the role of a change in charge at this location. Substitution of K for N at position 240 also reduced the single-channel conductance, although to a lesser extent (Fig. 9). This suggests that both the size of the side chain as well as its charge affects the conductance. We also mutated N240 to E. The conductance was increased over that of ROMK2, although again the effect was smaller than that of the R substitution (Fig. 9). Finally, we mutated the neighboring E239 residue to Q to subtract a negative charge from this part of the channel. This decreased the conductance slightly from 43 to 39 pS (unpublished data). Although the change in the net charge was the same as that of the N240R mutation, the decrease in conductance was smaller. Therefore, the precise position of the mutation, as well as the changes in charge and size, is important.


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

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

Effect of charge at position 240 on single-channel conductance. Conductances measured between −40 and −140 mV were ROMK2, 43 pS; C13, 31 pS; N240R, 31 pS; N240K, 37pS; and N240E, 45pS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234023&req=5

fig9: Effect of charge at position 240 on single-channel conductance. Conductances measured between −40 and −140 mV were ROMK2, 43 pS; C13, 31 pS; N240R, 31 pS; N240K, 37pS; and N240E, 45pS.
Mentions: To examine the mechanism of the effect of the N240R substitution on conductance, we tested for the role of a change in charge at this location. Substitution of K for N at position 240 also reduced the single-channel conductance, although to a lesser extent (Fig. 9). This suggests that both the size of the side chain as well as its charge affects the conductance. We also mutated N240 to E. The conductance was increased over that of ROMK2, although again the effect was smaller than that of the R substitution (Fig. 9). Finally, we mutated the neighboring E239 residue to Q to subtract a negative charge from this part of the channel. This decreased the conductance slightly from 43 to 39 pS (unpublished data). Although the change in the net charge was the same as that of the N240R mutation, the decrease in conductance was smaller. Therefore, the precise position of the mutation, as well as the changes in charge and size, is important.

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