Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

Extracellular Ca2+ controls outward rectification by apical cation channels in toad urinary bladder: Patch-clamp and whole-bladder studies

Summary Outward rectifying. cation channels were observed in the epithelial cells of the urinary bladder of the toad.Bufo marinus. As studied in isolated cells using the patch-clamp technique, the channel has an average conductance of 24 and 157 pS for pipette potentials between 0 and +60 mV and –60 to –100 mV, respectively, when the major cation in both bath and pipette solutions is K⁺. The conductance of the cannel decreasen with increasing dehydration energy of the permeant monovalent cation in the oder Rb⁺=K⁺>Na⁺>Li⁺. Reversal potentials near zero under biionic conditions imply that the permeabilities for all four of these cations are smiliar. The channel is sensitive to quinidine sulfate but not to amiloride. It shares several pharmacological and biophysical properties with an outwardly-rectifying, vasopressin-sensitive pical K⁺ conductive pathway described previously for the toad urinary bladder. We demonstrate, in both single-channel and whole-bladder studies, that the outward rectification is a consequence of interaction of the chanel with extracellular divalent cations, particularly Ca²⁺, which blocks inward but not outward current. Various divalent cations impart different degrees of outward rectification to the conductive pathway. Concentrations of Mg²⁺ and Ca²⁺ required for halfmaximal effect are 3×10^–4 and 10^–4 m, resopectively. For Co²⁺ the values are 10^–6 m at +50 mV and a 10^–4 m at +200 mV. The mechanism of blockade by divalent cations is not established, but does not seem to involve a voltage-dependent interaction in which the blocker penetrates the transmembrane electric field. In the absence of divalent cations in the mucosal solution, the magnitudes of inward current carried by Rb⁺, K⁺, Na⁺ and Li⁺ through the apical K⁺ pathway at any transepithelial voltage, are in the same order as in the single-channel studies. We propose that the cation channel observed by us in isolated epithelial cells is the single-channel correlate of the vasopressin-sensitive apical K⁺ conductive pathway in the toad urinary bladder and is also related to the oxytocin- and divalent cation-sensitive apical condictivity observed in frog skin and urinary bladder.     

Kir4.1 K+ channels are regulated by external cations

The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K⁺-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K⁺ concentrations ([K⁺]_o). We found that Kir4.1 is regulated by K⁺_o. Increased [K⁺]_o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K⁺ from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pH_i-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K⁺_o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs⁺ and Ba²⁺ substitute for K⁺ and prevent deactivation of the channel in the absence of K⁺_o. Cs⁺ blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb⁺ and NH₄⁺ permeate Kir4.1, only Rb⁺ is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel’s sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH₄⁺ to permeate the channel without inducing these changes.     

A Conserved Arginine Residue in the Pore Region of an Inward Rectifier K Channel (IRK1) as an External Barrier for Cationic Blockers

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg¹⁴⁸, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K⁺ > Rb⁺ > NH₄ ⁺ >> Na⁺) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg²⁺ and Ca²⁺, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg²⁺ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs⁺ and Ba²⁺, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg²⁺ or Ca²⁺ and that to the barrier peak for block by Cs⁺ or Ba²⁺ suggest that Arg¹⁴⁸ is located between the external blocking site for Mg²⁺ or Ca²⁺ and the deeper blocking site for Cs⁺ or Ba²⁺ in the IRK1 channel. It is concluded that Arg¹⁴⁸ serves as a barrier to cationic blockers, keeping Mg²⁺ and Ca²⁺ out from the electric field of the membrane.     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Voltage-dependent inhibition of outward Kir2.1 currents by extracellular spermine

Outward currents through inward rectifier Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells. Extracellular monovalent and divalent cations have been shown to reduce outward K⁺ conductance. In the present study, we examined whether spermine, with four positive charges, also inhibits outward Kir2.1 currents. We found that extracellular spermine inhibits steady-state outward Kir2.1 currents, an effect that increases as the voltage becomes more depolarizing, similar to that observed for intracellular spermine. However, several lines of evidence suggest that extracellular spermine does not inhibit outward currents by entering the cytoplasmic pore. Site-directed mutagenesis studies support that extracellular spermine directly interacts with the extracellular domain. In addition, we found that the voltage-dependent decay of outward Kir2.1 currents was necessary for inhibition by extracellular spermine. Further, a region at or near the selectivity filter and the cytoplasmic pore are involved in the voltage-dependent decay and thus in the inhibition of outward currents by extracellular spermine. Taken together, the data suggest that extracellular spermine bound to the mouth of the extracellular pore may induce an allosteric effect on voltage-dependent decay of outward currents, a process in which a region in the vicinity of the selectivity filter and cytoplasmic pore are involved. This study reveals that the extracellular pore domain, the selectivity filter and the cytoplasmic pore are in communication and this coupling is involved in modulating K⁺ conduction in the Kir2.1 channel.     

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

Modulation of P2X4 pore closure by magnesium,potassium, and ATP

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 μs we have investigated how metal-complexed ATP stabilizes the channel's open state and prevents its closing. We have identified two metal-binding sites, Mg²⁺ and potassium K⁺, one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP-binding site (MBS2), similar to those characterized in Gulf Coast P2X. Our data indicate that when Mg²⁺ and K⁺ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg²⁺ ions bound to the MBS2 impeded the collapse of the open-state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg²⁺ in the MBS2 was replaced with K⁺ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and the pore collapsed. This collapse was apparent when fewer than two ATPs were bound to MBS2 in the presence of K⁺. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study therefore provides structural insights into the differential modulation of ATP activation of P2X4 by Mg²⁺ and K⁺.     

Single-Channel Activities of the Human Epithelial Ca2+ Transport Proteins CaT1 and CaT2

The human epithelial channels, CaT1 and CaT2, were expressed in oocytes, and their single-channel characteristics were compared. In the presence of Na⁺ and K⁺ as charge carriers in the pipette solutions, channel activities were observed only when the the extracellular sides of the patches were exposed to nominally Ca²⁺- and Mg²⁺-free solutions. In patches of both CaT1- and CaT2-expressing oocytes, multiple channel openings were observed, but the current levels were higher in CaT2-expressing oocytes, particularly at more negative voltages. With K⁺ as a charge carrier in patches of CaT1-expressing oocytes, the channel activity was low at −10 to −60 mV, but increased dramatically at more negative potentials. This voltage dependence was observed in the presence of both Na⁺ and K⁺. The channel activity with Na⁺, however, was higher at all potentials. Differences between the voltage dependencies for the two cations were also observed in CaT2-expressing oocytes, but the channel activities were higher than those in CaT1-expressing oocytes, particularly in the presence of Na⁺. We also found that low concentrations of extracellular Mg²⁺ (5–50 μm) elicited a strong inhibitory action on the CaT channels. Activation of the CaT1 and CaT2 channels by hyperpolarization and other factors may promote increased Ca²⁺ entry that participates in stimulation of intestinal absorption and renal reabsorption and/or other Ca²⁺ transport mechanisms in epithelial cells. Received: 8 March 2001/Revised: 24 July 2001     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Susceptibility of the guard-cell K+-uptake channel KST1 to Zn2+ requires histidine residues in the S3-S4 linker and in the channel pore

 Potassium channels are inhibited by several mono- and divalent cations. To identify sites involved in the interaction between K⁺ channels and cationic effectors, we expressed the potato (Solanum tuberosum L.) guard-cell K⁺-uptake channel KST1 in Xenopus oocytes. This channel was reversibly blocked by extracellular Zn²⁺ in the micromolar range. In the presence of this heavy metal, steady-state currents were reduced in a pH-dependent but voltage-independent manner. Since Zn²⁺-inhibition was less effective at elevated external proton concentrations, we generated alanine mutants with respect to both extracellular histidines in KST1. Whereas substitution of the pore histidine H271 resulted in a reduced blockade by Zn²⁺, the channel mutant KST1-H160A in the S3-S4 linker lost most of its Zn²⁺ sensitivity. Since both histidines alter the susceptibility of KST1 to Zn²⁺, the block may predominantly result from these two sites. We thus conclude that the S3-S4 linker is involved in the formation of the outer pore. Received: 3 May 1999 / Accepted: 8 July 1999     

Multi-ion distributions in the cytoplasmic domain of inward rectifier potassium channels

Inward rectifier potassium (Kir) channels act as cellular diodes, allowing unrestricted flow of potassium (K⁺) into the cell while preventing currents of large magnitude in the outward direction. The rectification mechanism by which this occurs involves a coupling between K⁺ and intracellular blockers—magnesium (Mg²⁺) or polyamines—that simultaneously occupy the permeation pathway. In addition to the transmembrane pore, Kirs possess a large cytoplasmic domain (CD) that provides a favorable electronegative environment for cations. Electrophysiological experiments have shown that the CD is a key regulator of both conductance and rectification. In this study, we calculate and compare averaged equilibrium probability densities of K⁺ and Cl⁻ in open-pore models of the CDs of a weak (Kir1.1-ROMK) and a strong (Kir2.1-IRK) rectifier through explicit-solvent molecular-dynamics simulations in ∼1 M KCl. The CD of both channels concentrates K⁺ ions greater than threefold inside the cytoplasmic pore while IRK shows an additional K⁺ accumulation region near the cytoplasmic entrance. Simulations carried out with Mg²⁺ or spermine (SPM⁴⁺) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K⁺ in both channels. The results also show that SPM⁴⁺ behaves differently inside these two channels. Although SPM⁴⁺ remains inside the CD of ROMK, it diffuses around the entire volume of the pore. In contrast, this polyatomic cation finds long-lived conformational states inside the IRK pore, interacting with residues E224, D259, and E299. The strong rectifier CD is also capable of sequestering an additional SPM⁴⁺ at the cytoplasmic entrance near a cluster of negative residues D249, D274, E275, and D276. Although understanding the actual mechanism of rectification blockade will require high-resolution structural information of the blocked state, these simulations provide insight into how sequence variation in the CD can affect the multi-ion distributions that underlie the mechanisms of conduction, rectification affinity, and kinetics.     

Ionic interactions of Ba2+ blockades in the MthK K+ channel

The movement and interaction of multiple ions passing through in single file underlie various fundamental K⁺ channel properties, from the effective conduction of K⁺ ions to channel blockade by Ba²⁺ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba²⁺ with permeant ions within the ion conduction pathway of the MthK K⁺ channel. We found that, as typical of K⁺ channels, the MthK channel was blocked by Ba²⁺ at the internal side, and the Ba²⁺-blocking effect was enhanced by external K⁺. We also obtained crystal structures of the MthK K⁺ channel pore in both Ba²⁺–Na⁺ and Ba²⁺–K⁺ environments. In the Ba²⁺–Na⁺ environment, we found that a single Ba²⁺ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba²⁺–K⁺ environment, Ba²⁺ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba²⁺ blockade of K⁺ channels.     

Effects of extracellular calcium and protons on osteoclast potassium currents

During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca²⁺ and H⁺. We examined the effects of these cations on two types of K⁺ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mm Ca²⁺, pH 7.4), the voltage-gated, outwardly rectifying K⁺ current activates at approximately 45 mV and the conductance is half-maximally activated at –29 mV (V _0.5). Increasing [Ca²⁺]_out rapidly and reversibly shifted the current-voltage (I–V) relation to more positive potentials. Current at –29 mV decreased to 28 and 9% of control current at 5 and 10 mm [Ca²⁺]_out, respectively. This effect of elevating [Ca²⁺]_out was due to a positive shift of the K⁺ channel voltage activation range. Zn²⁺ or Ni²⁺ (5 to 500 m) also shifted the I–V relation to more positive potentials and had additional effects consistent with blockade of the K⁺ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K⁺ current, the potency sequence was Zn²⁺ > Ni²⁺ > Ca²⁺. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K⁺ current, changes in the concentration of extracellular H⁺ or Ca²⁺ did not shift the voltage activation range of the inwardly rectifying K⁺ current. These findings are consistent with Ca²⁺ and other cations affecting voltage-dependent gating of the osteoclast outwardly rectifying K⁺ channel through changes in surface charge.This work was supported by The Arthritis Society and the Medical Research Council of Canada. S.M.S. is supported by a Scientist Award and S.J.D. by a Development Grant from the Medical Research Council.     

Blockade of a Retinal cGMP-gated Channel by Polyamines

The cyclic nucleotide–gated (CNG) channel in retinal rods converts the light-regulated intracellular cGMP concentration to various levels of membrane potential. Blockade of the channel by cations such as Ca²⁺ and Mg²⁺ lowers its effective conductance. Consequently, the membrane potential has very low noise, which enables rods to detect light with extremely high sensitivity. Here, we report that three polyamines (putrescine, spermidine, and spermine), which exist in both the intracellular and extracellular media, also effectively block the CNG channel from both sides of the membrane. Among them, spermine has the greatest potency. Extracellular spermine blocks the channel as a permeant blocker, whereas intracellular spermine appears to block the channel in two conformations—one permeant, and the other non- (or much less) permeant. The membrane potential in rods is typically depolarized to approximately −40 mV in the dark. At this voltage, K _1/2 of the CNG channel for extracellular spermine is 3 μM, which is 100–1,000-fold higher affinity than that of the NMDA receptor-channel for extracellular spermine. Blockade of the CNG channel by polyamines may play an important role in suppressing noise in the signal transduction system in rods.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.