The S4-S5 loop contributes to the ion-selective pore of potassium channels

Mutagenesis experiments on voltage-gated K+ channels have suggested that the ion-selective pore is comprised mostly of H5 segments. To see whether regions outside of the H5 segment might also contribute to the pore structure, we have studied the effect of single amino acid substitutions in the segment that connects the S4 and S5 putative transmembrane segments (S4-S5 loop) on various permeation properties of Shaker K+ channels. Mutations in the S4-S5 loop alter the Rb+ selectivity, the single-channel K+ and Rb+ conductances, and the sensitivity to open channel block produced by intracellular tetraethylammonium ion, Ba2+, and Mg2+. The block of Shaker K+ channels by intracellular Mg2+ is surprising, but is reminiscent of the internal Mg2+ blockade of inward rectifier K+ channels. The results suggest that the S4-S5 loop constitutes part of the ion-selective pore. Thus, the S4-S5 loop and the H5 segment are likely to contribute to the long pore characteristic of voltage-gated K+ channels.     

Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca2+, but can be induced to carry a slowly declining Na+ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na+, PX/PNa, in order to estimate the diameter of the conducting pore. Ammonium (NH4+) exhibited the highest relative permeability (PNH4/PNa = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had PTMA/PNa of 0.09. N-methyl-D-glucamine (0.50 x 0.64 x 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH4+ reduced the slow decline of monovalent current ("inactivation") upon lowering [Ca2+]o. This kinetic effect of extracellular NH4+ can be accounted for by an increase in intracellular pH (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa of 6.8. In several channel types, Mg2+ has been shown to produce rectification by a voltage-dependent block mechanism. Mg2+ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs+ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg2+ contributes to inward rectification by blocking in a voltage-dependent manner, with a z delta product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with z delta of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca2+ channels, achieves selectivity for Ca2+ by selective binding in a large pore with current-voltage characteristics shaped by internal Mg2+.     

High selectivity of the i(f) channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarization-activated inward current (i(f)) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K+ pipette, replacement of 90% external Na+ by Li+ caused a -24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs+ pipette, the i(f) current decreased almost proportionally to the decrease in external [Na+]o as Li+ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG+) substitution, suggesting that the relative permeability of Li+ compared with Na+ for the i(f) channel is as low as that of NMG+. When Cs+ or Rb+ was substituted for internal K+, the fully activated I/V relationship for i(f) showed strong inward rectification with a positive reversal potential, indicating low permeability of the i(f) channel for Cs+ and Rb+. These results show that the i(f) channel is highly selective for Na+ and K+ and will not pass the similar ions Li+ and Rb+. Such a high degree of selectivity is unique and may imply that the structure of the i(f) channel differs greatly from that of other Na+ and K+ conducting channels.     

Alveolar epithelial fluid clearance persists in the presence of moderate left atrial hypertension in sheep

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from -160 to +80 mV with a constant opening probability (Po). Although Po is low ( approximately 0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive Po seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.     

Selectivity changes during activation of mutant Shaker potassium channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in approximately 40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of approximately 1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.     

Structure-function study on a de novo synthetic hydrophobic ion channel

Ion conduction properties of a de novo synthesized channel, formed from cyclic octa-peptides consisting of four alternate L-alanine (Ala) and N'-acylated 3-aminobenzoic acid (Aba) moieties, were studied in bilayer membranes. The single-channel conductance was 9 pS in symmetrical 500 mM KCl. The channel favored permeation of cations over anions with a permeability ratio (PCl-/PK+) of 0.15. The selectivity sequence among monovalent cations based on permeability ratio (PX+/PK+) fell into an order: NH4+(1.4) > Cs+(1. 1) >/= K+(1.0) > Na+(0.4) > Li+(0). The conductance-activity relationship of the channel in K+ solutions followed simple Michaelis-Menten kinetics with a half-maximal saturating activity of 8 mM and a maximal conductance of 9 pS. The permeability ratio PNa+/PK+ remained constant ( approximately 0.40) under biionic concentrations from 10 to 500 mM. These results suggests that the channel is a one-ion channel. The pore diameter probed by a set of organic cations was approximately 6 A. The single-channel current was blocked by Ca2+ in a dose-dependent manner that followed a single-site titration curve with a voltage-dependent dissociation constant of 0.6 mM at 100 mV. The electric distance of the binding site for Ca2+ was 0.07 from both entrances of the channel, indicating the presence of two symmetrical binding sites in each vicinity of the channel entrance. Correlations between conduction properties and structural aspects of the channel are discussed in terms of a three-barrier and two-binding-site (3B2S) model of Eyring rate theory. All available structural information supported an idea that the channel was formed from a tail-to-tail associated dimer of the molecule, the pore of which was lined with hydrophobic acyl chains. This is the first report to have made a systematic analysis of ion permeation through a hydrophobic pore.     

Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.     

Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation.     

A novel oestrogen-regulated gene in human breast cancer cells identified by differential display

Shaker K+ channels inactivate through two distinct molecular mechanisms: N-type, which involves the N-terminal domain and C-type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C-type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C-type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C-type inactivated channels. The structural changes Shaker channels undergo during C-type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.     

Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme

Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.     

External barium block of Shaker potassium channels: evidence for two binding sites

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.     

Short periods of hypoxia activate a K+ current in central neurons

The effect of hypoxia on ionic currents was studied in acutely dissociated substantia nigra neurons. Using an external solution containing 0 mM Na+ and 0.5 mM Co2+, we found that overall whole-cell outward currents increased by 15-20% during 3-4 min of hypoxia. This current was voltage sensitive and could be completely blocked by TEA- and Cs+, suggesting that this is a K+ current. In cell-attached patches with 150 mM K+ in the external solution, we recorded a large-conductance outward current which was not observed during baseline and was reversibly activated by hypoxia. These results therefore provide the first direct evidence for the activation of K+ channels during O2 deprivation in central neurons.     

Respiration and ion permeability of the inner membrane in rat "sodium" liver mitochondria

Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes.     

Analysis of leak current properties in the lobster stretch receptor neurone

Experiments were performed to characterize the so-called leak current of the slowly adapting stretch receptor neurone of the European lobster with respect to its ionic basis, its kinetics and its pharmacology. Estimates of the leak current were obtained by subtraction of a Na-K pump current and of an unspecific impalement current from a non-dynamic ('instantaneous') current, recorded in a voltage range from approximately -120 to approximately -30 mV, after blockage of spike-generating currents and a hyperpolarization-activated inwardly rectifying current (Q-current). The leak current, estimated in this way, was seen to reverse direction at the cell's K+ equilibrium voltage, thus indicating that it is carried by K+ passing through channels which, also, proved to be permeable to Rb+ and NH4+, but not permeable to Na+ or Cl- to any significant extent. Kinetically, the leak current was found to be characterized by being enhanced by increases in extracellular K+ and by being subject to outward rectification, most distinctly at elevated extracellular [K+]. In quantitative terms, these kinetic properties could be accounted for by a mathematical model comprising (1) a one-site two-barrier Eyring formulation describing ion permeation through membrane channels and (2) an ordinary dose-response relationship describing the channel-opening effect of K+ at an extracellular regulatory site. Pharmacologically, the leak current proved to be distinguished by being reversibly blockable, in a non-voltage dependent manner, by CO2+ (Kd = 0.9 mM, Hill coefficient 1.1) and procaine, but not by Ba2+, Gd3+, bupivacaine (a local anesthetic), or other K+ channel blockers such as TEA, 4-AP and Cs+. It is concluded that, in native unimpaled cells, the K+ carried leak current (1) is setting the resting voltage together with the (mainly) Na(+)-carried Q-current and the Na-K pump current, (2) is determining the cell's firing threshold, together with the spike generating currents, and (3) is also stabilizing the cell's membrane excitability in conditions of varying extracellular [K+], by virtue of its K+ sensitivity.     

A novel type of internal barium block of a maxi-K+ channel from human vas deferens epithelial cells

We have recently shown that a maxi-K+ channel from vas deferens epithelial cells contains two Ba2+-binding sites accessible from the external side: a "flickering" site located deep in the channel pore and a "slow" site located close to the extracellular mouth of the channel. Using the patch-clamp technique, we have now studied the effect of internal Ba2+ on this channel. Cytoplasmic Ba2+ produced a voltage- and concentration-dependent "slow" type of block with a dissociation constant of approximately 100 microM. However, based on its voltage dependence and sensitivity to K+ concentration, this block was clearly different from the external "slow" Ba2+ block previously described. Kinetic analysis also revealed a novel "fast flickering" block restricted to channel bursts, with an unblocking rate of approximately 310 s(-1), some 10-fold faster than the external "flickering" block. Taken together, these results show that this channel contains multiple Ba2+-binding sites within the conduction pore. We have incorporated this information into a new model of Ba2+ block, a novel feature of which is that internal "slow" block results from the binding of at least two Ba2+ ions. Our results suggest that current models for Ba2+ block of maxi-K+ channels need to be revised.     

Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis

The structurally well-characterized scorpion toxin Agitoxin2 inhibits ion permeation through Shaker K+ channels by binding to the external pore entryway. Scanning mutagenesis identified a set of inhibitor residues critical for making energetic contacts with the channel. Using thermodynamic mutant cycle analysis, we have mapped channel residues relative to the known inhibitor structure. This study constrains the position of multiple channel residues within the pore-forming loops; in one stretch, we have been able to map five out of seven contiguous residues to the inhibitor interaction surface, including those involved in ion selectivity. One interaction in particular, that of K27M on the inhibitor with Y445F on the channel, is unique in that it depends on the K+ ion concentration. These results reveal a shallow vestibule formed by the pore loops at the K+ channel entryway. The selectivity filter is located at the center of the vestibule close to (approximately 5 A) the extracellular solution.     

Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mM. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 microM) and by removal of extracellular Cl-. These results strongly suggest that ammonium enters the cell by contransport of NH4+ with Cl-. Removal of extracellular Na+ did not abolish the NH(4+)-induced acidification. The NH(4+)-induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mM Ba2+ showing that NH4+ did not enter through Ba(2+)-sensitive ion channels. Application of 2 mM NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mM NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl-. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)-Cl- cotransporter described in kidney cells.     

Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K+. TASK-2 currents are blocked by quinine (IC50 = 22 microM) and quinidine (65% of inhibition at 100 microM) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6. 5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.     

Hydrophobic mutations alter the movement of Mg2+ in the pore of voltage-gated potassium channels

The permeation pathways of the voltage-gated K+ channels Kv3.1 and ShakerB delta 6-46 (ShB delta) were studied using Mg2+ block. Internal Mg2+ blocked both channels in a voltage-dependent manner, and block was partially relieved by external K+, consistent with Mg2+ binding within the pore. The kinetics of Mg2+ block was much faster for Kv3.1 than for ShB delta. Fast block of Kv3.1 was transferred to ShB delta with transplantation of the P-region, but not of S6. The difference in the P-region, causing the change in Mg2+ binding kinetics, was attributed to ShB delta (V443) and its analog Kv3.1(L401), because in both channels leucine at this position gave fast block, whereas valine gave slow block. For Kv3.1 the major determinant of the voltage dependence of Mg2+ binding resided primarily in the off rate, whereas for Kv3.1(L401V) the voltage dependence resided primarily in the on rate, consistent with a change in the rate-limiting barrier for Mg2+ binding. Our data suggest that hydrophobic residues at positions 401 of Kv3.1 and 443 of ShB delta act as barriers to the movement of Mg2+ in the pore.