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Abstract
Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.
Figure 2. Competitive inhibition of polyamine block by polyamine toxins. (A) Representative traces from the same giant excised inside-out patch showing block of wild-type Kir2.1 channel by various concentrations of spermine (S) in the absence (left) or presence (right) of 30 nM philanthotoxin (T). Recording conditions and voltage-clamp protocol as in Fig. 1. (BâD) Doseâresponse of outward currents at +40 mV (after 200 ms) to spermine in Kir2.1 wild type (B), D172N (C), and E224G (D), in the absence (â) or presence (â¢) of philanthotoxin. The gray crosses show the normalized data for philanthoxin + spermine, to better illustrate the shift in K0.5. The superimposed curves are least-squares fits to the Hill equation. Hill coefficients ranged from 0.7 to 0.9. (E) Doseâresponse curves for wild-type Kir2.1 currents to spermidine (Spd) in the absence (â) and presence (â¢, gray crosses, normalized) of 30 nM philanthoxin. Hill coefficients ranged from 0.42 to 0.51. (F) Doseâresponse curve of wild-type Kir2.1 currents at +40 mV to spermine in the absence (â) or presence (â¢, gray crosses, normalized) of 10 nM argiotoxin (AT). Hill coefficients ranged from 0.77 to 0.99. Data points in BâF represent the mean ± SEM for n = 4â6 patches.
Figure 3. Long pore plugging model of inward rectification. (A) Reaction scheme for the âlong poreâ model of direct open channel block by spermine (S) proposed by Lopatin et al. (1995) (shaded area), modified to include direct competitive block by philanthotoxin (T). Cartoons illustrate states in which the channel pore is open (O), blocked by spermine (B1âB3), philanthotoxin (B4), or both spermine and philanthotoxin (B5). K1â5 represent equilibrium constants (kreverse/kforward) for the various transitions. See text for details. (BâC) Fits of the direct block model in A to the experimental data for the spermineâphilanthotoxin interaction in wild-type Kir2.1 shown in Fig. 3 B, where â are spermine alone, ⢠are spermine + philanthotoxin, and gray crosses are normalized spermine + philanthotoxin. Model parameters in B: K1 = 7.75 à 10â5 M, K2 = 7.75 à 10â5 M, K3 = 7.75 à 10â5 M, K4 = 47 à 10â9 M, K5 = 47 à 10â9 M. In C, the B5 state was disabled by increasing K5 to 103 M. In neither case could the model reproduce quantitatively the increase in K0.5 for spermine in the presence of philanthotoxin.
Figure 4. Time course of inactivation of outward currents in wild-type Kir2.1 channels. (A) Macroscopic currents were recorded from a giant inside-out patch excised from a Xenopus oocytes expressing wild-type Kir2.1 channels, while superfusing with Mg and polyamine-free bath solution. At the times indicated after patch excision, outward wild-type Kir2.1 currents elicited by voltage clamp pulses to +60 mV from a holding potential of â30 mV showed progressively slower inactivation, reaching a steady state after 5 min. Despite identical amplitudes of inward current at â30 mV (indicating complete unblock), endogenous polyamines still remained available to reblock the current upon depolarization to +30 mV, albeit at a slower rate (e.g., 1- vs. 3-min traces). (B) Rate of inactivation of outward current in four patches. Fraction of noninactivating current (Ifinal/Ipeak) at the end of a 60-ms voltage clamp to +40 mV is plotted for each patch. The rate of inactivation reached steady state by 5 min of washout. (C) Hypothetical schema of channel block by polyamines (left) or polyamine toxins (right) using an intrinsic gate mechanism. Unlike spermine, it is assumed that the hydrophobic head of the toxin molecule is too large to block the pore when its polyamine end is tethered to the intrinsic gate. Conjectured locations of two negatively charged residues D172 and E224 are shown at the pore docking site and at the polyamine-binding site on the intrinsic gate, respectively.
Figure 5. Intrinsic gate model of inward rectification. (A) The simplest reaction scheme showing spermine (S) and philanthotoxin (T) competition for an intrinsic gate and a pore-docking site in the Kir2.1 channel. Cartoons illustrate the blocked states (B1âB7) and the O3 state in which the toxin is bound to the intrinsic gate, preventing spermine's access to its high affinity blocking pathway (O1 â O2 â B1). K1â5 represent equilibrium constants (kreverse/kforward) for the various transitions. Shaded area represents states involved in spermine block in the absence of toxin, see Fig. 3 A for comparison. See text for further details. (BâD) Fits of the intrinsic gate model in A to the experimental data for the spermineâphilanthotoxin interaction shown in Fig. 3, BâD, where â are spermine alone, ⢠are spermine + philanthotoxin, and gray crosses are normalized spermine + philanthotoxin. Model parameters for wild-type Kir2.1 (B): K1 = 7.75 à 10â5 M, K2 = 7.75 à 10â5 M, K3 = 5.8 à 10â9 M, K4 = 6 à 10â9 M, K5 = 47 à 10â 9M; for D172N (C): K1 = 7.75 à 10â5 M, K2 = 115 à 10â5 M, K3 = 132 à 10â9 M, K4 = 89 à 10â6 M, K5 = 1,220 à 10â9 M; for E224G (D): K1 = 103 M, K2 = 103 M, K3 = 103 M, K4 = 6,030 à 10â9 M, K5 = 7,090 à 10â9 M.
Abrams,
The role of a single aspartate residue in ionic selectivity and block of a murine inward rectifier K+ channel Kir2.1.
1996, Pubmed
Abrams,
The role of a single aspartate residue in ionic selectivity and block of a murine inward rectifier K+ channel Kir2.1.
1996,
Pubmed
Aleksandrov,
Inward rectification of the IRK1 K+ channel reconstituted in lipid bilayers.
1996,
Pubmed
,
Xenbase
Brackley,
Selective antagonism of native and cloned kainate and NMDA receptors by polyamine-containing toxins.
1993,
Pubmed
,
Xenbase
Donevan,
Multiple actions of arylalkylamine arthropod toxins on the N-methyl-D-aspartate receptor.
1996,
Pubmed
Doupnik,
The inward rectifier potassium channel family.
1995,
Pubmed
Doyle,
The structure of the potassium channel: molecular basis of K+ conduction and selectivity.
1998,
Pubmed
Fakler,
Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine.
1995,
Pubmed
,
Xenbase
Ficker,
Spermine and spermidine as gating molecules for inward rectifier K+ channels.
1994,
Pubmed
,
Xenbase
Ho,
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
1989,
Pubmed
Lopatin,
Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification.
1994,
Pubmed
,
Xenbase
Lopatin,
The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines.
1995,
Pubmed
,
Xenbase
Matsuda,
Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+.
,
Pubmed
Oliva,
The mechanism of rectification of iK1 in canine Purkinje myocytes.
1990,
Pubmed
Shieh,
Inward rectification of the IRK1 channel expressed in Xenopus oocytes: effects of intracellular pH reveal an intrinsic gating mechanism.
1996,
Pubmed
,
Xenbase
Stanfield,
The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+.
1994,
Pubmed
Taglialatela,
Specification of pore properties by the carboxyl terminus of inwardly rectifying K+ channels.
1994,
Pubmed
,
Xenbase
Taglialatela,
C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.
1995,
Pubmed
,
Xenbase
Vandenberg,
Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions.
1987,
Pubmed
Wible,
Gating of inwardly rectifying K+ channels localized to a single negatively charged residue.
1994,
Pubmed
,
Xenbase
Williams,
Effects of Agelenopsis aperta toxins on the N-methyl-D-aspartate receptor: polyamine-like and high-affinity antagonist actions.
1993,
Pubmed
,
Xenbase
Yang,
Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel.
1995,
Pubmed
,
Xenbase
