Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca2+ and K+ channels

Protein toxins from venomous animals exhibit remarkably specific and selective interactions with a wide variety of ion channels. Hanatoxin and grammotoxin are two related protein toxins found in the venom of the Chilean Rose Tarantula, Phrixotrichus spatulata. Hanatoxin inhibits voltage-gated K⁺ channels and grammotoxin inhibits voltage-gated Ca²⁺ channels. Both toxins inhibit their respective channels by interfering with normal operation of the voltage-dependent gating mechanism. The sequence homology of hanatoxin and grammotoxin, as well as their similar mechanism of action, raises the possibility that they interact with the same region of voltage-gated Ca²⁺ and K⁺ channels. Here, we show that each toxin can interact with both voltage-gated Ca²⁺ and K⁺ channels and modify channel gating. Moreover, mutagenesis of voltage-gated K⁺ channels suggests that hanatoxin and grammotoxin recognize the same structural motif. We propose that these toxins recognize a voltage-sensing domain or module present in voltage-gated ion channels and that this domain has a highly conserved three-dimensional structure.     

Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca²⁺ channels, the pore-forming, antagonist-binding α₁ subunit, considerably less is understood about how β subunits contribute to neuronal Ca²⁺ channel function. We studied the role of the Ca²⁺ channel β₃ subunit, the major Ca²⁺ channel β subunit in neurons, by using a gene-targeting strategy. The β₃ deficient (β₃−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β₃−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca²⁺ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β₃ subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β₃−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca²⁺ channels. The reduction in the expression of N-type Ca²⁺ channels in the β₃−/− mice may be expected to impair Ca²⁺ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca²⁺-dependent K⁺ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca²⁺ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca²⁺ influx. To determine the Ca²⁺ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K⁺, Na⁺, or Ca²⁺. For modest concentrations of a single permeant cation, Ca²⁺ carried much more receptor current than did either K⁺ or Na⁺. For higher cation concentrations, however, the flux of Na⁺ or K⁺ through the transduction channel exceeded that of Ca²⁺. For mixtures of Ca²⁺ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca²⁺ over monovalent cations but also that Ca²⁺ carries substantial current even at low Ca²⁺ concentrations. At physiological cation concentrations, Ca²⁺ flux through transduction channels can change the local Ca²⁺ concentration in stereocilia in a range relevant for the control of adaptation.     

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca²⁺ currents evoke synaptic transmitter release. Of six types of Ca²⁺ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca²⁺ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca²⁺ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca²⁺ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca²⁺ influx during a presynaptic action potential. This Ca²⁺ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca²⁺ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca²⁺ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.     

Molecular basis of plant-specific acid activation of K+ uptake channels

During stomatal opening potassium uptake into guard cells and K⁺ channel activation is tightly coupled to proton extrusion. The pH sensor of the K⁺ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K⁺ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K⁺ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K⁺ uptake channels.     

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B₂ bradykinin and M₁ muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP₃ receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP₃ to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca²⁺]_i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca²⁺]_i were blocked by depletion of Ca²⁺ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca²⁺ from IP₃-sensitive Ca²⁺ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Ca2+-sensitive inactivation of L-type Ca2+ channels depends on multiple cytoplasmic amino acid sequences of the α1C subunit

Ca²⁺-dependent inactivation of Ca²⁺ currents is a physiological phenomenon widely associated with L-type Ca²⁺ channels. Although the pore-forming α_1C subunit of the channel is the target for Ca²⁺ binding, the amino acid sequences involved in the binding and/or in the coordination of Ca²⁺-dependent inactivation are still unclear. Based on previous experiments, we have prepared truncation mutants of a human α_1C subunit by systematically deleting an EF-hand motif and sequences in a segment of 80 amino acids in the carboxyl-terminal tail. We found that the rate as well as the Ca²⁺ dependence of inactivation of currents through these mutated channels were very different. We have identified three amino acid sequences, the presence of which is important for Ca²⁺-dependent inactivation: (i) a putative Ca²⁺-binding EF-hand motif, (ii) two hydrophilic residues (asparagine and glutamic acid) 77–78 amino acids downstream of the EF-hand motif, and (iii) a putative IQ calmodulin binding motif. We suggest that Ca²⁺-dependent inactivation is a cooperative process involving several amino acid sequences in cytoplasmic segments of the α_1C subunit.     

Effects of extracellular Ca2+ concentration on hair-bundle stiffness and gating-spring integrity in hair cells

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca²⁺ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca²⁺ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca²⁺ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca²⁺ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca²⁺ concentration; the maximal stiffness of ≈1200 μNm⁻¹ occurred when bundles were bathed in solutions containing 250 μM Ca²⁺, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca²⁺ concentration below ≈100 nM, hair-bundle stiffness fell to ≈200 μNm⁻¹ and no longer exhibited Ca²⁺-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca²⁺ concentration.     

Hair cell-specific splicing of mRNA for the α1D subunit of voltage-gated Ca2+ channels in the chicken’s cochlea

The L-type voltage-gated Ca²⁺ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca²⁺-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type α_1D subunit that predominates in hair cells of the chicken’s cochlea. The α_1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the α_1D mRNA contributes to the unusual behavior of the hair cell’s voltage-gated Ca²⁺ channels.     

Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida

The sperm acrosome reaction is a Ca²⁺-dependent exocytotic event that is triggered by adhesion to the mammalian egg’s zona pellucida. Previous studies using ion-selective fluorescent probes suggested a role of voltage-sensitive Ca²⁺ channels in acrosome reactions. Here, whole-cell patch clamp techniques are used to demonstrate the expression of functional T-type Ca²⁺ channels during mouse spermatogenesis. The germ cell T current is inhibited by antagonists of T-type channels (pimozide and amiloride) as well as by antagonists whose major site of action is the somatic cell L-type Ca²⁺ channel (1,4-dihydropyridines, arylalkylamines, benzothiazapines), as has also been reported for certain somatic cell T currents. In sperm, inhibition of T channels during gamete interaction inhibits zona pellucida-dependent Ca²⁺ elevations, as demonstrated by ion-selective fluorescent probes, and also inhibits acrosome reactions. These studies directly link sperm T-type Ca²⁺ channels to fertilization. In addition, the kinetics of channel inhibition by 1,4-dihydropyridines suggests a mechanism for the reported contraceptive effects of those compounds in human males.     

Killing K Channels with TEA+

Tetraethylammonium (TEA⁺) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K⁺ and TEA⁺ irreversibly decreased the potassium current when there was no K⁺ outside. Five minutes of perfusion with 20 mM TEA⁺, followed by removal of TEA⁺, reduced potassium current to <5% of its initial value. The irreversible disappearance of K channels with TEA⁺ could be prevented by addition of ≥ 10 mM K⁺ to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA⁺. Killing is much less evident when an axon is held at −110 mV to tightly close all of the channels. The longer-chain TEA⁺ derivative decyltriethylammonium (C10⁺) had irreversible effects similar to TEA⁺. External K⁺ also protected K channels against the irreversible action of C10⁺. It has been reported that removal of all K⁺ internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K⁺ within the pore is required to maintain function. Our evidence further suggests that the crucial location for K⁺ binding is external to the (internal) TEA⁺ site and that TEA⁺ prevents refilling of this location by intracellular K⁺. Thus in the absence of extracellular K⁺, application of TEA⁺ (or C10⁺) has effects resembling dekalification and kills the K channels.     

Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca²⁺ release by using a technique of imaging local Ca²⁺ release, or “Ca²⁺ spikes”, at subcellular sites; and we tracked the kinetics of Ca²⁺ release triggered by L-type Ca²⁺ channels. At 0 mV, Ca²⁺ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca²⁺ channels with the Ca²⁺ channel agonist, FPL64176, did not prolong or trigger secondary Ca²⁺ spikes, even though two-thirds of the sarcoplasmic reticulum Ca²⁺ remained available for release. Latency of Ca²⁺ spikes coincided with the first openings but not with the reopenings of L-type Ca²⁺ channels. After an initial maximal release, even a multi-fold increase in unitary Ca²⁺ current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca²⁺ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca²⁺ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca²⁺-activated K⁺ currents (I_K[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in I_K[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ngml⁻¹. β1-neuregulin treatment for 12 hr also confers Ca²⁺ sensitivity onto large-conductance (285 pS) K⁺ channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca²⁺ currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Predominance of the α1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken’s cochlea

The voltage-gated Ca²⁺ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca²⁺ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca²⁺ channels, we investigated the expression of L-type α₁ subunits in hair cells of the chicken’s cochlea. In PCRs with five different pairs of degenerate primers, we always obtained α_1D products, but only once an α_1C product and never an α_1S product. A full-length α_1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the α_1D subunit was about 90% identical to those of the mammalian α_1D subunits. In situ hybridization confirmed that the α_1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the α_1D mRNA is 100–500 times more abundant than the α_1C mRNA. We conclude that most, if not all, voltage-gated Ca²⁺ channels in hair cells contain an α_1D subunit. Furthermore, we propose that the α_1D subunit plays a hitherto undocumented role at tonic synapses.     

Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α_1A subunit of P/Q-type Ca²⁺ channels have different patterns of interactions with synaptic proteins. The BI isoform of α_1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca²⁺ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α_1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α_1A is Ca²⁺-dependent, with maximum affinity at 10–20 μM Ca²⁺. Although the rbA isoform of α_1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca²⁺-dependent. These differential, Ca²⁺-dependent interactions of Ca²⁺ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca²⁺ influx through these channels.     

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K⁺ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K⁺ currents, an inactivating outward-rectifying K⁺ current was characterized (plant A-type current: I_AP). I_AP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. I_AP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward I_AP was mainly carried by K⁺ ions. In contrast to the sustained outward-rectifying K⁺ currents, cytosolic alkaline pH was found to inhibit I_AP and extracellular K⁺ was required for I_AP activity. Furthermore, increasing cytosolic free Ca²⁺ in the physiological range strongly inhibited I_AP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K⁺ current in a higher plant cell. Regulation of I_AP by diverse factors including membrane potential, cytosolic Ca²⁺ and pH, and extracellular K⁺ and Ca²⁺ implies that the inactivating I_AP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.     

Voltage dependence of the pattern and frequency of discrete Ca2+ release events after brief repriming in frog skeletal muscle

Applying a brief repolarizing pre-pulse to a depolarized frog skeletal muscle fiber restores a small fraction of the transverse tubule membrane voltage sensors from the inactivated state. During a subsequent depolarizing test pulse we detected brief, highly localized elevations of myoplasmic Ca²⁺ concentration (Ca²⁺ “sparks”) initiated by restored voltage sensors in individual triads at all test pulse voltages. The latency histogram of these events gives the gating pattern of the sarcoplasmic reticulum (SR) calcium release channels controlled by the restored voltage sensors. Both event frequency and clustering of events near the start of the test pulse increase with test pulse depolarization. The macroscopic SR calcium release waveform, obtained from the spark latency histogram and the estimated open time of the channel or channels underlying a spark, exhibits an early peak and rapid marked decline during large depolarizations. For smaller depolarizations, the release waveform exhibits a smaller peak and a slower decline. However, the mean use time and mean amplitude of the individual sparks are quite similar at all test depolarizations and at all times during a given depolarization, indicating that the channel open times and conductances underlying sparks are essentially independent of voltage. Thus, the voltage dependence of SR Ca²⁺ release is due to changes in the frequency and pattern of occurrence of individual, voltage-independent, discrete release events.     

Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K⁺, Na⁺ and Ca²⁺ channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K⁺ channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K⁺ (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.