The polyamine binding site in inward rectifier K+ channels

Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the "rectification controller" residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly "locked" into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the "rectification controller" residue, near the extracellular entrance to the channel.     

The role of the cytoplasmic pore in inward rectification of Kir2.1 channels

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a "long-pore" effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.     

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.     

Localization of the pH gate in Kir1.1 channels

We used cysteine-modifying reagents to localize the pH-sensitive gate in the renal inward-rectifier K(+) channel Kir1.1a (ROMK1). Cytoplasmic-side methanethiosulfonate (MTS) reagents blocked K(+) permeation in native Kir1.1 channels, expressed in Xenopus oocytes. Replacement of three cysteines in the N-terminus, C-terminus, and transmembrane domains eliminated this sensitivity to MTS reagents, as measured with inside-out macropatches. Reintroduction of one cysteine at 175-Kir1.1a in the second transmembrane domain allowed blockade of the open channel by the MTS reagents MTSEA, MTSET, and MTSES and by Ag(+). However, closure of the channel by low pH protected it from modification. Cysteine was also introduced into position G223, which is thought to line the cytoplasmic pore of the channel. MTSET blocked G223C in both the open and closed state. In contrast, MTSEA reduced G223C single-channel conductance from 40 to 23 pS but did not produce complete block. We conclude that cytoplasmic acidification induces a conformational change in the channel protein that prevents access of cysteine-modifying reagents, and presumably also K(+) ions, to the transmembrane pore from the cytoplasm. This is consistent with localization of the Kir1.1 pH gate at the helix bundle crossing near the cytoplasmic end of the transmembrane pore.     

Interaction mechanisms between polyamines and IRK1 inward rectifier K+ channels

Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding-unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.     

Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels

Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.     

Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis

Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.     

Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.

The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.     

Ser165 in the second transmembrane region of the Kir2.1 channel determines its susceptibility to blockade by intracellular Mg2+

The strong inward rectification of Kir2.1 currents is reportedly due to blockade of the outward current by cytoplasmic magnesium (Mg(2+)(i)) and polyamines, and is known to be determined in part by three negatively charged amino acid residues: Asp172, Glu224, and Glu299 (D172, E224, E299). Our aim was to identify additional sites contributing to the inward rectification of Kir2.1 currents. To accomplish this, we introduced into wild-type Kir2.1 and its D172N and D172N & E224G & E299S mutants various point mutations selected on the basis of a comparison of the sequences of Kir2.1 and the weak rectifier sWIRK. By analyzing macroscopic currents recorded from Xenopus oocytes using two-electrode voltage clamp, we determined that S165L mutation decreases inward rectification, especially with the triple mutant. The susceptibility to blockade by intracellular blockers was examined using HEK293 transfectants and the inside-out patch clamp configuration. The sensitivity to spermine was significantly diminished in the D172N and triple mutant, but not the S165L mutant. Both the S165L and D172N mutants were less susceptible to blockade by Mg(2+)(i) than the wild-type channel, and the susceptibility was still lower in the D172N & S165L double mutant. These results suggest that S165 is situated deeper into the pore from inside than D172, where it is accessible to Mg(2+)(i) but not to spermine. The single channel conductance of the D172N mutant was similar to that of the wild-type Kir2.1, whereas the conductance of the S165L mutant was significantly lower. Permeation by extracellular Rb+ (Rb(+)(o)) was dramatically increased by S165L mutation, but was increased only slightly by D172N mutation. By contrast, the Rb+/K+ permeability ratio was increased equally by D172N and S165L mutation. We therefore propose that S165 forms the narrowest part of the Kir2.1 pore, where both extracellular and intracellular blockers plug the permeation pathway.     

Evidence for sequential ion-binding loci along the inner pore of the IRK1 inward-rectifier K+ channel

Steep rectification in IRK1 (Kir2.1) inward-rectifier K(+) channels reflects strong voltage dependence (valence of approximately 5) of channel block by intracellular cationic blockers such as the polyamine spermine. The observed voltage dependence primarily results from displacement, by spermine, of up to five K(+) ions across the narrow K(+) selectivity filter, along which the transmembrane voltage drops steeply. Spermine first binds, with modest voltage dependence, at a shallow site where it encounters the innermost K(+) ion and impedes conduction. From there, spermine can proceed to a deeper site, displacing several more K(+) ions and thereby producing most of the observed voltage dependence. Since in the deeper blocked state the leading amine group of spermine reaches into the cavity region (internal to the selectivity filter) and interacts with residue D172, its trailing end is expected to be near M183. Here, we found that mutation M183A indeed affected the deeper blocked state, which supports the idea that spermine is located in the region lined by the M2 and not deep in the narrow K(+) selectivity filter. As to the shallower site whose location has been unknown, we note that in the crystal structure of homologous GIRK1 (Kir3.1), four aromatic side chains of F255, one from each of the four subunits, constrict the intracellular end of the pore to approximately 10 A. For technical simplicity, we used tetraethylammonium (TEA) as an initial probe to test whether the corresponding residue in IRK1, F254, forms the shallower site. We found that replacing the aromatic side chain with an aliphatic one not only lowered TEA affinity of the shallower site approximately 100-fold but also eliminated the associated voltage dependence and, furthermore, confirmed that similar effects occurred also for spermine. These results establish the evidence for physically separate, sequential ion-binding loci along the long inner pore of IRK1, and strongly suggest that the aromatic side chains of F254 underlie the likely innermost binding locus for both blocker and K(+) ions in the cytoplasmic pore.     

Gating dependence of inner pore access in inward rectifier K(+) channels

Cation channel gating may occur either at or below the inner vestibule entrance or at the selectivity filter. To differentiate these possibilities in inward rectifier (Kir) channels, we examined cysteine accessibility in the ATP-gated Kir6.2 channel. MTSEA and MTSET both block channels and modify M2 cysteines with identical voltage dependence. If entry is restricted to open channels, modification rates will slow in ATP-closed channels, but because the reagent can be trapped in the pore following brief openings, this may not be apparent until open probability is extremely low (<0.01). When these conditions are met, modification does slow significantly, indicating gated access and highlighting an important caveat for interpretation of MTS-accessibility measurements: reagent "trapping" in nominally "closed" channels may obscure gated access.     

Serotonin and cocaine-sensitive inactivation of human serotonin transporters by methanethiosulfonates targeted to transmembrane domain I

To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4 degrees C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.     

The external TEA binding site and C-type inactivation in voltage-gated potassium channels

The location of the tetraethylammonium (TEA) binding site in the outer vestibule of K+ channels, and the mechanism by which external TEA slows C-type inactivation, have been considered well-understood. The prevailing model has been that TEA is coordinated by four amino acid side chains at the position equivalent to Shaker T449, and that TEA prevents a constriction that underlies inactivation via a foot-in-the-door mechanism at this same position. However, a growing body of evidence has suggested that this picture may not be entirely correct. In this study, we reexamined these two issues, using both the Kv2.1 and Shaker potassium channels. In contrast to results previously obtained with Shaker, substitution of the tyrosine at Kv2.1 position 380 (equivalent to Shaker 449) with a threonine or cysteine had a relatively minor effect on TEA potency. In both Kv2.1 and Shaker, modification of cysteines at position 380/449 by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) proceeded at identical rates in the absence and presence of TEA. Additional experiments in Shaker demonstrated that TEA bound well to C-type inactivated channels, but did not interfere with MTSET modification of C449 in inactivated channels. Together, these findings rule out the possibility that TEA binding involves an intimate interaction with the four side chains at the position equivalent to Shaker 449. Moreover, these results argue against the model whereby TEA slows inactivation via a foot-in-the-door mechanism at position 449, and also argue against the hypothesis that the position 449 side chains move toward the center of the conduction pathway during inactivation. Occupancy by TEA completely prevented MTSET modification of a cysteine in the outer-vestibule turret (Kv2.1 position 356/Shaker position 425), which has been shown to interfere with both TEA binding and the interaction of K+ with an external binding site. Together, these data suggest that TEA is stabilized in a more external position in the outer vestibule, and does not bind via direct coordination with any specific outer-vestibule residues.     

Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence approximately 5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QA(C10)) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider ( approximately 6 A) than the ion selectivity filter ( approximately 3 A). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QA(C10) should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.     

The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu > delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.     

Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels

Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody-induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 microM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel-PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.     

Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.     

Block of the Kir2.1 Channel Pore by Alkylamine Analogues of Endogenous Polyamines

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K⁺ channels by entering deeply into a long, narrow pore, displacing K⁺ ions to the outside of the membrane, with this displacement of K⁺ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K⁺ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.     

Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis

In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca(2+)-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 A) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 A) resulted in modification rates 10(3)-10(4) faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg(+), and Ag(+) appeared poorly state dependent, whereas modification rates by MTSET were 10(3) faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg(+), and Ag(+), arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K(+) ions for the closed channel configuration.