- Top of page
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 ∼ spermine ≈ 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.
Inward rectifier K+ channels (Kir) conduct inward currents at potentials more negative than the reversal potential of K+, but permit much smaller currents at potentials positive than the reversal potential (Hille, 2001). Under physiological conditions, the mechanism underlying this inward rectification is the voltage (membrane potential, Vm)-dependent blockade of outward K+ currents by both intracellular Mg2+ (Matsuda et al. 1987; Vandenberg, 1987) and polyamines binding to the aspartate at site 172 (D172; Lu & MacKinnon, 1994; Stanfield et al. 1994; Wible et al. 1994), the glutamate at site 224 (E224; Taglialatela et al. 1995; Yang et al. 1995) and the glutamate at site 299 (E299; Kubo & Murata, 2001). D172 has been shown to provide a strong energetic contribution to spermine and Mg2+ binding in the pore of the Kir2.1 channel (Lopatin et al. 1994; Stanfield et al. 1994; Wible et al. 1994) and thus it is generally considered to be a binding site, or to be located close to the binding site for these internal blockers. However, it is unclear to what extent a large molecule such as spermine occupies space within the pore.
Kir channels are integral membrane proteins that consist of two transmembrane segments (M1 and M2) flanking a pore-forming loop (P), and N- and C-terminal cytoplasmic domains (Fig. 1A). Recently, it has been shown that the M2 segment of a prokaryotic inward rectifier K+ channel, KirBac1.1, is a helix, as determined by X-ray crystallography (Kuo et al. 2003). Site-directed mutagenesis and cysteine scanning studies, have also shown that the M2 segments of several eukaryotic Kir channels are also helical (Choe et al. 1995; Minor et al. 1999; Loussouarn et al. 2000). However, other studies show that the sequences in the M2 segment do not have obvious periodicity corresponding to α helices or β strands (Collins et al. 1997; Lu et al. 1999). Scanning cysteine accessibility studies have revealed that several amino acids in the M2 segment of the cloned Kir2.1 channel line the inner pore of the channel. According to the secondary structure (Kubo et al. 1993), the amino end of the M2 segment is located externally to the carboxyl end. Yet, the relative positions of the amino acids of the M2 segment during a functional state remain unknown.
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.
Download figure to PowerPoint
In this study we employed the spermine bound at D172 to determine the relative positions of the pore-lining residues of the M2 segment (Fig. 1B). We rationalized that if amino acids are located externally to the spermine bound to D172, then their accessibility to internally applied trimethylammonioethylmethane thiosulphonate (MTSET) will be abolished by spermine. On the other hand, for those amino acids positioned internally to the spermine bound to D172 and located far enough from it, their accessibility to MTSET will remain unaffected by spermine. In addition, since spermine occupies a range of spaces and carries charges, the accessibility of MTSET to the amino acids adjacent to the spermine binding site will be somewhat reduced, through physical and/or electrostatic repulsion. In addition to D172, spermine also induces inward rectification through interaction with E224 and E299. Therefore, we also examined the effects of spermine on the accessibility of cysteine residues introduced in the channels with a neutral residue (asparagine or cysteine) replacing the aspartate at site 172. We provide evidence that the inward rectification induced by the spermine bound to E224 and E299 is not due to a direct occlusion of the cytoplasmic pore in the Kir2.1 channel. Possible mechanisms underlying the involvement of E224 and E299 in inward rectification are discussed.