The effects of spermine on the accessibility of residues in the M2 segment of Kir2.1 channels expressed in Xenopus oocytes

Abstract

We examined the effects of spermine binding to aspartate at site 172 on the accessibility of internal trimethylammonioethylmethane thiosulphonate (MTSET) to substituted cysteines within the pore of a Kir2.1 channel. Spermine prevented MTSET modification in Q164C and G168C mutants, indicating that sites 164 and 168 are located externally to the spermine binding site. The rates of MTSET modification were significantly reduced by spermine in I176C mutants, indicating that site 176 is located internally to D172 and that the bound spermine hinders the reaction of MTSET with cysteine at site 176. Spermidine, putrescine and Mg2+ also decreased MTSET modification at site 176. The order of effect is putrescine > spermidine approximately = spermine approximately = Mg2+. To account for the electrostatic and physical repulsion between MTSET and polyamines, possible locations of polyamines in the pore are discussed. In D172C mutants, the spermine that bound to sites 224 and 299 completely inhibited channels at +40 mV, yet MTSET remained accessible to site 172. In addition, in the D172C mutant, spermine did not affect the exit rate of Ba2+ bound to the threonine at the site 141. These results indicate that spermine bound at the cytoplasmic pore induces channel closure at positions 141-172. The effects of spermine on the accessibility of amino acids in the pore may shed light on the structural and functional relationships of the Kir2.1 channels during inward rectification.

Figure 1. Schematic plot of a Kir2.1 subunit

A, secondary structure of a Kir2.1 subunit. B, amino acid sequence of the M2 segment of the Kir2.1 subunit. Asterisks mark the residues that were mutated into cysteines and were accessible to MTSET.

Figure 2. Effects of spermine on MTSET modification in the D172C mutant

A and B, current traces in the absence and (solid lines) and presence (dotted lines) of MTSET (2 mm) mutant in the control (Ctrl) and 1 µm spermine (Spm), respectively. The number of seconds below each dotted line indicates the time after MTSET treatment. The time courses of MTSET modification in the control and spermine are shown in the lower panels. The horizontal lines indicate the zero current levels.

Figure 3. Effects of spermine on MTSET modification in various cysteine mutants

A–G, the time courses of MTSET modification in the indicated cysteine mutants in the control and spermine (1 µm). The horizontal lines indicate the time span of MTSET (2 mm) treatment.

Figure 4. Effects of spermine on MTSET modification in the V169C/D172N mutant

Each panel illustrates the time course of MTSET modification in the control (left panel) and in spermine (right panel).

Figure 5. Effects of spermine on MTSET modification in the I176C/D172N mutant

Each panel illustrates the time courses of MTSET modification in the control (left panel) and in spermine (right panel).

Figure 7. Effects of polyamines and Mg²⁺ on MTSET modification in the I176C/E224G/E299S mutant

A–E, the time course of MTSET modification in the control solution and the indicated blocker.

Figure 6. Effects of spermine on MTSET modification in the D172C/E224G/E299S and I176C/D172N/E224G/E299S mutants

A and B, current traces in the absence and (solid lines) and presence (dotted lines) of MTSET (2 mm) in the D172C/E224G/E299S and I176C/D172N/E224G/E299S mutants, respectively, exposed to the control solution (left panel) and spermine-added solution (right panel). The corresponding time course of MTSET modification is shown below the traces.

Figure 8. Effects of spermine on the recovery from Ba²⁺ block in the D172C mutant

A, the two-pulse voltage protocol. The time interval between the two identical pulses was increased by 15 ms between sweeps (starting with 10 ms) to assess recovery from the block induced by 10 µm extracellular Ba²⁺. B and C, currents traces recorded in the control and in spermine (1 µm), respectively. D, time courses of recovery from Ba²⁺ block in the control (•) and 1 µm spermine (○), respectively. Continuous lines are the fit of the data to a monoexponential function.

Figure 9. Proposed interaction of polyamines with the Kir2.1 channel

A, the molecular structures and dimensions of polyamines. Atom C is plotted in grey and atom N in blue. The dimensions of polyamines were determined by ChemIDPlus (http://chem.sis.nlm.nih.gov/chemidplus/). B, residues 49-98 of one MthK subunit. The colour codes are: blue, A58 (T141); red F87 (D172); green, V91 (I176). The structure is taken from Protein Data Bank (code 1lnq). A spermine molecule is fitted in the pore cavity with the second amine aligned horizontally with F87 (D172). Spd = spermidine, Put = putrescine