On the Theory of Ion Transport Across the Nerve Membrane, II. Potassium Ion Kinetics and Cooperativity (with x = 4)

We use a tetrahedral model of four interacting protein subunits to represent the K⁺ channel or gate in the squid nerve membrane. The kinetic predictions, with varying degrees of cooperativity, are compared with experimental observations, especially those of Hodgkin and Huxley (J. Physiol. 117, 500, 1952) and of Cole and Moore (Biophys. J. 1, 1, 1960). The tentative conclusion reached is that if there is any cooperativity present it must be rather weak. There is no indication here that cooperativity improves the Hodgkin-Huxley assumption of independent “subunits”. Other related models will be discussed in Part III. We also find evidence against the suggestion that there is cooperativity between K⁺ channels arranged in patches of a two-dimensional lattice.     

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

Mutations in the K_V7.2 gene encoding for voltage-dependent K⁺ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S₄ domain of K_V7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K_V7.2 and/or K_V7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K_V7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K_v7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K⁺ channel impairment, and set the preclinical basis for the potential use of K_v7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K_V7.2 encephalopathy.     

Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state

C-type inactivation of K⁺ channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K⁺ channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K⁺ or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K⁺ channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d-Alanine. Protein semisynthesis was used to introduce d-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K⁺ channel K_vAP. For semisynthesis of the K_vAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and K_vAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.The ability of K⁺ channels to selectively conduct K⁺ ions is accomplished by a structural unit called the selectivity filter (1). The selectivity filter consists of four K⁺ binding sites built using the main chain carbonyl oxygens and the threonine side chain from the protein sequence, which is typically T-V-G-Y-G (Fig. 1A) (2, 3). This sequence, referred to as the signature sequence, is highly conserved among K⁺ channels (2). The high degree of conservation of the signature sequence indicates a similar structure for the selectivity filter of all K⁺ channels, which is in fact observed in the K⁺ channel structures presently available (4).Open in a separate windowFig. 1.The conductive and constricted conformations of the K⁺ selectivity filter. (A) Close-up view of the selectivity filter of wild-type KcsA channel at high K⁺ concentration [K⁺] (PDB ID code: 1k4c). Two diagonally opposite subunits are shown in stick representation. K⁺ ions are shown as purple spheres. (B) Macroscopic currents of the wild-type KcsA channel elicited by a pH jump show inactivation. Currents were elicited at +100 mV by a rapid change of solution pH, at the arrow, from pH 7.5 (10 mM Hepes-KOH, 200 mM KCl) to pH 3.0 (10 mM succinate, 200 mM KCl). The selectivity filter of the KcsA channels at low [K⁺] (C, PDB ID code: 1k4d) and in the 32-Ǻ open structure (D, PDB ID code: 3f5w) show the constricted conformation. A rotation of the Val76–Gly77 bond causes constriction of the pore. The Gly77 Cα–Cα distance in the opposite subunits is 8.1 Å for the conductive conformation and 5.4–5.5 Å for the constricted conformation at low [K⁺] or in the 32-Å open state. (E) Structure of the selectivity filter of KcsA^G77dA at high [K⁺] (PDB ID code: 2ih3). (F) A hypothetical structure of the KcsA^G77dA selectivity filter in the constricted conformation. Two adjacent subunits are shown. The methyl side chain of d-Ala77 of one subunit and the carbonyl oxygen atoms of the Val76 and d-Ala77 in the adjacent subunit that clash are shown in van der Waals (VDW) representation. (G) Structure of the selectivity filter of KcsA^G77dA at low [K⁺] (PDB ID code: 2ih1). (H) Superposition of the selectivity filter of the KcsA^G77dA in high [K⁺] (blue) and low [K⁺] (red) shows that the d-Ala substitution in the selectivity filter blocks the constricted conformation.In addition to ion discrimination, the selectivity filter participates in a gating process referred to as C-type inactivation, during which the channel transitions from the conductive state to a nonconductive state (5). C-type inactivation has been extensively investigated in voltage-gated K⁺ (K_v) channels and is observed on prolonged opening of K_v channels by a sustained membrane depolarization (4, 6). C-type inactivation is an effective mechanism to control K_v channel activity and to regulate action-potential frequency in an excitable cell (7). An inactivation process, which is similar to C-type inactivation, is also observed in K⁺ channels that do not belong to the K_v family, such as the bacterial K⁺ channel KcsA. The KcsA channel is gated by pH (8). A decrease in the intracellular pH causes channel opening by conformational changes at the bundle crossing of the pore lining helices. In the closed state, the bundle crossing of the pore lining helices acts as a barrier for the movement of ions across the membrane (9). Activation of the KcsA channel is followed by inactivation during which the current decreases (Fig. 1B) (10, 11). Inactivation in the KcsA channel is proposed to be C-type as it shares a number of functional similarities with C-type inactivation in K_v channels (12–14). This similarity, coupled with the amenability of KcsA to structural studies, has made it an attractive system for elucidating the structure of the selectivity filter in the C-type inactivated state.Models for the selectivity filter in the C-type inactivated state have been proposed based on structures of the KcsA channel at low K⁺ or in the open state. The selectivity filter of the KcsA channel undergoes a conformational change from the conductive state at high K⁺ to a nonconductive state at low K⁺ (Fig. 1C) (3, 15). In the low K⁺ conformation, there is a rotation around the Gly77–Val76 peptide bond that causes the α-carbon of Gly77 to twist inwards and constrict the pore. This rotation disrupts the second and third ion binding sites in the selectivity filter and renders the channel nonconductive (Fig. 1 A, C, and D). As the rate of C-type inactivation increases at low K⁺, the conformation of the selectivity filter at low K⁺ was proposed to represent the C-type inactivated state (16). Recently, a series of structures with varying degrees of opening at the bundle crossing of the pore lining helices were obtained by using a constitutively open mutant of the KcsA channel (17). Higher degrees of opening at the bundle crossing (25–32 Å) were accompanied by a conformational change in the selectivity filter that was presumed to be nonconductive (Fig. 1D). This nonconductive conformation of the selectivity filter was proposed to represent the C-type inactivated state. The conformations of the selectivity filter in low K⁺ or in the open-channel structure are quite similar except for slight differences toward the lower half of the selectivity filter and the orientation of the Thr75 side chain. Due to their similarity, we jointly refer to these conformations as the “constricted” conformation of the selectivity filter. Changes in the conformation of the KcsA selectivity filter at low K⁺ or low pH have also been detected by solution and solid-state NMR and are consistent with the constricted conformation of the selectivity filter (18–20).However, does the constricted conformation represent the selectivity filter in the C-type inactivated state? An important caveat of the structural studies is that the C-type inactivated state must be accurately captured by the conditions used for structure determination. Experimental validation is therefore necessary before the constricted conformation can be conclusively assigned as the C-type inactivated state. Here, we used unnatural amino acid mutagenesis to test whether the constricted conformation of the selectivity filter of the KcsA channel corresponds to the C-type inactivated state. We also used unnatural amino acid mutagenesis on the archaebacterial K_v channel K_vAP, to test whether the constricted conformation is relevant during C-type inactivation in a K_v channel. We show that inactivation in the K_vAP channel is functionally similar to C-type inactivation in a eukaryotic K_v channel. To carry out unnatural amino acid mutagenesis, we developed a modular semisynthesis of the K_vAP channel that allowed us to use chemical synthesis to modify the selectivity filter. Our results on the KcsA and the K_vAP channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.     

Setting the chaperonin timer: the effects of K+ and substrate protein on ATP hydrolysis

The effects of potassium ion on the nested allostery of GroEL are due to increases in the affinity for nucleotide. Both positive allosteric transitions, TT-TR and TR-RR, occur at lower [ATP] as [K⁺] is increased. Negative cooperativity in the double-ringed system is also due to an increase in the affinity of the trans ring for the product ADP as [K⁺] is increased. Consequently, (i) rates of ATP hydrolysis are inversely proportional to [K⁺] and (ii) the residence time of GroES bound to the cis ring is prolonged and the hemicycle time extended. Substrate protein suppresses negative cooperativity by decreasing the affinity of the trans ring for ADP, reducing the hemicycle time to a constant minimum. The trans ring thus serves as a variable timer. ATP added to the asymmetric GroEL-GroES resting-state complex lacking trans ring ADP is hydrolyzed in the newly formed cis ring with a presteady-state burst of ≈6 mol of Pi per mole of 14-mer. No burst is observed when the trans ring contains ADP. The amplitude and kinetics of ATP hydrolysis in the cis ring are independent of the presence or absence of encapsulated substrate protein and independent of K⁺ at concentrations where there are profound effects on the linear steady-state rate. The hydrolysis of ATP by the cis ring constitutes a second, nonvariable timer of the chaperonin cycle.     

Potassium channels: Structure-function relationships, diversity, and pharmacology

There are many different types of potassium (K⁺) channels: A good number are voltage-dependent, others are activated by variations of intracellular concentrations of Ca²⁺ and the activity of others is controlled by cytoplasmic variations of the ATP/ADP ratio or by variations of intracellular Na⁺ or arachidonic acid and other fatty acids; a large number are modulated by phosphorylation and/or interaction with G proteins. Considerable progress has been made in the past few years in the molecular knowledge of some of these channels. Some of the voltage-dependent K⁺ channels have been cloned. In each tissue several genes encode several different K⁺ channel subunits that assemble to form large families of voltage-dependent K⁺ channels with different biophysical properties (different voltage dependence, different time course), which are associated with different physiological functions. The molecular structure of other types of K⁺ channels is not yet solved. Investigation of the molecular pharmacology of K⁺ channels has also made tremendous progress recently. High-affinity ligands are now available for some of the voltage-dependent K⁺ channels, Ca²⁺-activated K⁺ channels, and ATP-sensitive K⁺ channels.     

Modulation of Kv Channel α/β Subunit Interactions

Voltage-gated K⁺ channels comprise the largest and most diverse class of ion channels. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle, as well as being the targets of several antiarrhythmic drugs in the heart. The multiplicity of Kv channel function is further enhanced through modulation by accessory β subunits, which confer rapid inactivation, alter current amplitudes, and promote cell surface expression. In addition, α/β interactions are also influenced by second messenger pathways. Recent evidence demonstrates that phosphorylation of Kv channel α and/or β subunits may dramatically affect channel properties. The functional response of different K⁺ channel subunits to activation of protein kinases represents not only a means to modulate subunit interactions, but also another mechanism for K⁺ channel diversity in vivo.     

Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome) caused by mutations in KCNJ10

We describe members of 4 kindreds with a previously unrecognized syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemia, metabolic alkalosis, and hypomagnesemia). By analysis of linkage we localize the putative causative gene to a 2.5-Mb segment of chromosome 1q23.2–23.3. Direct DNA sequencing of KCNJ10, which encodes an inwardly rectifying K⁺ channel, identifies previously unidentified missense or nonsense mutations on both alleles in all affected subjects. These mutations alter highly conserved amino acids and are absent among control chromosomes. Many of these mutations have been shown to cause loss of function in related K⁺ channels. These findings demonstrate that loss-of-function mutations in KCNJ10 cause this syndrome, which we name SeSAME. KCNJ10 is expressed in glia in the brain and spinal cord, where it is believed to take up K⁺ released by neuronal repolarization, in cochlea, where it is involved in the generation of endolymph, and on the basolateral membrane in the distal nephron. We propose that KCNJ10 is required in the kidney for normal salt reabsorption in the distal convoluted tubule because of the need for K⁺ recycling across the basolateral membrane to enable normal activity of the Na⁺-K⁺-ATPase; loss of this function accounts for the observed electrolyte defects. Mice deficient for KCNJ10 show a related phenotype with seizures, ataxia, and hearing loss, further supporting KCNJ10's role in this syndrome. These findings define a unique human syndrome, and establish the essential role of basolateral K⁺ channels in renal electrolyte homeostasis.     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James     

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K⁺) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K⁺ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K⁺ channel inactivation and exhibits a remarkable structural plasticity that correlates to K⁺ channel inactivation. The transmembrane helix 1 unwinds when the K⁺ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K⁺ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K⁺ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K⁺ channel pore.     

Initial steps of inactivation at the K+ channel selectivity filter

K⁺ efflux through K⁺ channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel’s selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K⁺ channel. Either Mg²⁺ or Ca²⁺ can reduce K⁺ efflux through MthK channels. However, Ca²⁺, but not Mg²⁺, can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca²⁺ ions can partially dehydrate, enabling selective accessibility of Ca²⁺ to a site at the entry to the selectivity filter. Ca²⁺ binding at the site interacts with K⁺ ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.Potassium (K⁺) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K⁺ efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K⁺ channels are further controlled by a gating phenomenon known as C-type inactivation, in which K⁺ conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K⁺ channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that C-type inactivation is associated with a conformational change near the external mouth of the K⁺ channel pore (i.e., at the canonical K⁺ channel selectivity filter) (3–11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K⁺ (6). Thus, K⁺ presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg²⁺, Ca²⁺, and Sr²⁺) as probes of inactivation mechanisms in MthK, a model K⁺ channel of known structure (Fig. 1) (12–14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our electrophysiological experiments indicate that, although each of these divalent metal ions can reduce the size of single channel currents, only Ca²⁺ and Sr²⁺ can enhance inactivation, whereas Mg²⁺ does not. Using molecular simulation and potential of mean force (PMF) calculations, we find that Ca²⁺, but not Mg²⁺, can shed its hydration shell waters to access a site, termed S5, at the entry to the channel’s selectivity filter (Fig. 1C) after displacement of K⁺ ions to the extracellular side of the channel. Subsequent dissociation of a K⁺ ion from the filter, in turn, favors a conformational change within the selectivity filter, contributing to enhanced inactivation. These results support a working hypothesis that directly relates dissociation of K⁺ with a structural change in the selectivity filter to initiate inactivation of K⁺ channels.Open in a separate windowFig. 1.Structure and activation properties of MthK. (A) Presumed biological structure of MthK shown as a Cα-trace [Protein Data Bank (PDB) ID code 3RBZ]. The channel consists of a transmembrane pore domain tethered to a ring of RCK domains, which mediate channel activation by cytoplasmic Ca²⁺ (green spheres). The gray-shaded region represents the presumed plasma membrane; dashed lines represent the linker region between the pore and RCK gating ring that is unresolved in the crystal structure. (B) High-resolution structure of the MthK pore domain, with the selectivity filter shown in ball and stick representation (PDB ID code 3LDC). Subunits in the front and back have been removed for clear visualization of the conduction pathway (inside dashed rectangle), with K⁺ ions shown as purple spheres and ordered water molecules shown as red spheres. (C) –Magnified view of the MthK conduction pathway (boxed region in B) with potential ion binding sites (S0–S_cav) indicated. (D) Po vs. [Ca²⁺] (black symbols) and [Cd²⁺] (red symbols) from currents recorded at −100 mV. MthK activation requires ∼20-fold lower [Cd²⁺] compared with [Ca²⁺]. Curves represent fits with a Hill equation with the following parameters: EC₅₀ = 1.0 mM and n_H = 9.5 for Ca²⁺; EC₅₀ = 49 μM and n_H = 8.4 for Cd²⁺. (E) Representative single channel currents from reconstituted MthK at depolarized voltages with 200 mM KCl at both sides of the membrane and Ca²⁺ or Cd²⁺ at the cytoplasmic side of the channel as indicated. Cd²⁺ can fully activate MthK at concentrations that produce much less fast blockade than Ca²⁺. O and C indicate open and closed current levels, respectively. (F) Unitary current vs. voltage for MthK channels activated with 30 and 100 μM Cd²⁺ (green and red, respectively) and 2 mM Ca²⁺ (black). Smooth curves are drawn for display only; 100 μM Cd²⁺ results in nominal levels of fast blockade, yielding large outward current.     

Influence of potassium levels on one-year outcomes in elderly patients with acute heart failure

BackgroundAbnormal serum potassium levels (K⁺) in patients with heart failure (HF) relate to worse prognosis. We evaluated whether admission K⁺ levels predict 1-year outcomes in elderly patients admitted for acute HF.MethodsWe evaluated 2865 patients aged >74 years from the RICA Spanish Heart Failure Registry, classified according to admission serum K⁺ levels: hyperkalemia (>5.5 mmol/L), normokalemia (3.5–5.5 mmol/L) and hypokalemia (<3.5 mmol/L). We explored whether K⁺ levels were significantly associated with one-year all-cause mortality or hospital readmission and their combination.ResultsMean admission K⁺ value was 4.3 ± 0.6 mmol/L; 97 patients (3.38%) presented with hyperkalemia and 174 (6.06%) with hypokalemia. Overall, 43% of the patients died or were readmitted for HF during the follow-up period; the risk was higher for those with hyperkalemia (59% vs 41% in hypokalemic patients). The HR for one-year mortality was 1.43 (p = .073) and 1.67 for readmissions (p = .007) when K⁺ was >5.5 mmol/L and 1.08 (p = .618) and 0.90 (p = .533) respectively for K⁺ < 3.5 mmol/L. The HR for the combined outcome was 1.59 (1.19–2.13); p = .002 in hyperkalemic patients and 0.96 (0.75–1.23); p = .751in hypokalemic patients. Multivariate analysis showed a significant association of admission K⁺ values >5.5 mmol/L with the combined outcome of mortality and readmission (HR 1.15 [95% CI 1.04–1.27], p = .008).ConclusionIn patients hospitalized for decompensated HF, admission hyperkalemia predicts a higher mid-term risk for HF readmission and mortality, probably related to the significant higher risk of readmission.     

Filter gate closure inhibits ion but not water transport through potassium channels

The selectivity filter of K⁺ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K⁺ hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K⁺. Here, we show that K⁺ channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K⁺ channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p_f of (6.9 ± 0.6) × 10⁻¹³ cm³⋅s⁻¹ for both mutants. “Collapse” of the selectivity filter upon K⁺ removal did not alter p_f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K⁺-containing medium to which K⁺-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K⁺ in the aqueous solution, which indicates an effective K⁺ dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K⁺ ions in the selectivity filter act to mutually destabilize binding.     

Pancreatic β-cell K<Subscript>ATP</Subscript> channels: Hypoglycaemia and hyperglycaemia

The pancreatic β-cell ATP-sensitive K⁺ channel (K_ATP channel) plays a critical role in glucose homeostasis by linking glucose metabolism to electrical excitability and insulin secretion. Changes in the intracellular ratio of ATP/ADP mediate the metabolic regulation of channel activity. The β-cell K_ATP channel is a hetero-octameric complex composed of two types of subunits: four inward-rectifying potassium channel pore-forming (Kir6.2) subunits and four high-affinity sulfonylurea receptor 1 (SUR1) subunits. Kir6.2 and SUR1 are encoded by the genes KCNJ11 and ABCC8, respectively. Mutations in these genes can result in congenital hyperinsulinism and permanent neonatal diabetes. This review highlights the important role of the β-cell K_ATP channel in glucose physiology and provides an introduction to some of the other review articles in this special edition of the Reviews in Endocrine and Metabolic Disorders.     

Potassium secretion in rat distal colon during dietary potassium loading: role of pH regulated apical potassium channels

Background—Chronicdietary K⁺ loading increases the abundance of largeconductance (210 pS) apical K⁺ channels in surface cells ofrat distal colon, resulting in enhanced K⁺ secretion inthis epithelium. However, the factors involved in the regulation ofthese K⁺ channels are at present unclear.
Aims—To evaluate theeffect of dietary K⁺ loading on intracellular pH and itsrelation to large conductance apical K⁺ channel activity insurface cells of rat distal colon.
Methods/Results—Asassessed by fluorescent imaging, intracellular pH was higher inK⁺ loaded animals (7.48 (0.09)) than in controls (7.07 (0.04); p<0.01) when surface cells were bathed in NaCl solution, and asimilar difference in intracellular pH was observed when cells werebathed in Na₂SO₄ solution (7.67 (0.09) and 6.92 (0.05) respectively; p<0.001). Ethylisopropylamiloride (EIPA; aninhibitor of Na⁺-H⁺ exchange; 1 µM) decreasedintracellular pH when surface cells from K⁺ loaded animalswere bathed in either solution, although the decrease was greater whenthe solution contained NaCl (ΔpH 0.50 (0.03)) rather thanNa₂SO₄ (ΔpH 0.18 (0.02); p<0.05). Incontrast, EIPA had no effect in cells from control animals. As assessedby patch clamp recording techniques, the activity of large conductance K⁺ channels in excised inside-out membrane patches fromdistal colonic surface cells of K⁺ loaded animals increasedtwofold when the bath pH was raised from 7.40 to 7.60. As assessed bycell attached patches in distal colonic surface cells fromK⁺ loaded animals, the addition of 1 µM EIPA decreasedK⁺ channel activity by 50%, consistent with reversal ofNa⁺-H⁺ exchange mediated intracellular alkalinisation.
Conclusion—Intracellularalkalinisation stimulates pH sensitive large conductance apicalK⁺ channels in rat distal colonic surface cells as part ofthe K⁺ secretory response to chronic dietary K⁺loading. Intracellular alkalinisation seems to reflect an increase inEIPA sensitive Na⁺-H⁺ exchange, which may be amanifestation of the secondary hyperaldosteronism associated with thismodel of colonic K⁺ adaptation.

     

Selectivity filter ion binding affinity determines inactivation in a potassium channel

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Ion permeation gating within the selectivity filter (SF) of potassium (K⁺) channels has been proposed to control channel activity in different ways for different family members. In voltage-dependent K⁺ (K_V) channels, the SF has been proposed to underlie C-type inactivation (1–3), resulting in the progressive loss of current following the activation of a channel gate located near the intracellular side of the pore (4). C-type inactivation in K_V channels has been shown to be strongly dependent on the affinity of a particular binding site for permeant ions in the pore, and the affinity of these pore sites has been proposed to depend not only on the SF chemical composition, but also on regions outside of the SF (5–8). A structure-function model for this mechanism has been provided most specifically by studies of the proton-gated KcsA channel (9–11) where opening of the activation gate is correlated with a conformational constriction and a decrease in ion occupancy within the four K⁺ binding sites of the SF (named S1 to S4) (9, 12–15). Experimental and structural studies of KcsA in low K⁺ showed that the SF constriction consists of an outward flip of the carbonyl groups of the Gly77, in the middle of the signature sequence (TVGYG) of K⁺ channels, associated with a loss of K⁺ binding at site S2 of the SF (9, 14, 16–20). These conformational changes were accompanied by the binding of several water molecules behind the SF, stabilizing this constricted (also called flipped) state by sterically preventing the SF from switching back into its conductive state (16, 18–20). While some reports challenge this view (21, 22), this activation gate-coupled collapse of the SF is now generally accepted as the mechanism underlying C-type inactivation in K⁺ channels.Several types of ligand-dependent K⁺ channels, including those opened by binding Ca²⁺, such as the BK and MthK channels (23–27), do not exhibit traditional C-type inactivation, despite possessing an identical SF with K_V and KcsA channels. Furthermore, these channels have been proposed to actually gate at the SF (28–32) although a recent cryogenic electron microscopy (cryo-EM) structure of MthK in the absence of calcium (33) revealed a steric closure at the bundle crossing inner gate region, suggesting that there may be two gates involved in calcium gating. Nevertheless, at this time, the structural correlates of SF gating and the difference from inactivation are unknown.In the present study, we set out to first investigate whether we can capture different gating states of MthK by obtaining X-ray structures of its pore (Fig. 1A) in wide-ranging concentrations of K⁺. MthK channels have been previously shown to display a decrease in activity with depolarization, which is further augmented when external K⁺ concentration is lowered, a signature of SF gating and a hallmark of C-type inactivation (34). We reasoned that K⁺ titration of MthK pore structures may provide insights into the molecular causes for K⁺-dependent SF gating and will indicate whether it shares features with the C-type inactivation observed in KcsA (such as a collapsed SF). Unlike KcsA, MthK SF did not collapse in similarly low K⁺ concentrations, suggesting that the clue to why MthK does not display traditional C-type inactivation may lie in understanding the molecular underpinnings that contribute to SF conformational change. Thus, we next investigated the dependence of SF conformation on ion occupancy and used molecular dynamics (MD) simulations to reveal a uniquely low affinity central S2 site in MthK, which may play a lead role in the SF-based channel closure. Overall, our results illustrate how the exact same sequence and structure of the SF in a K⁺ channel can lead to slight variations in K⁺ binding site chemistry, which in turn can lead to distinct functional phenotypes.Open in a separate windowFig. 1.Structure of the MthK pore in different K⁺ concentrations. (A) Overall architecture of wild-type MthK pore structure (three subunits of the tetrameric pore shown for clarity) crystallized with 150 mM K⁺. The SF is highlighted by dashed lines. Alignment of this structure with that crystallized in 6 mM K⁺ yields an all-atom root-mean-square deviation (RMSD) value of 0.25 Å. (B) K⁺-omit electron density maps (2F_o – F_c contoured at 2.0 σ) for SF atoms from two opposing subunits. Structures were solved in 150, 50, 11, and 6 mM [K⁺], as indicated. Crystallographic statistics are in SI Appendix, Table S1. (C) MD system with the MthK (ribbons) embedded in a lipid bilayer (gray sticks) bathed in 200 mM KCl (K⁺ as green spheres, Cl⁻ as blue spheres, and water as red and white sticks).     

Hypokalemia in rats produces resistance to dysrhythmias under halothane anesthesia

The arrnytnmogemc tnresnoia was investigates curing acute and chronic hypokalemia under halothane anesthesia with an epinephrine challenge in the rat model. It was hypothesized that in the setting of severe hypokalemia, general anesthesia would be arrhythmogenic and would be exaggerated with increased levels of catecholamines. Rats were divided into four groups as follows: normokalemic control (group I, n = 10), acute hypokalemia with furosemide (group II, n = 16), acute hypokalemia with hyperventilation (group III, n = 18), and chronic hypokalemia induced by a low potassium (K⁺) diet (group IV, n = 22). There were no significant differences (P < .05) in baseline K⁺ and arterial blood gases among the four groups. There was a significant difference between groups I and II and groups I and IV (P < 0.05) in serum K⁺ values after the low K⁺ diet, but no differences were observed between groups II and IV or groups I and 111 in serum K⁺ levels. There was no significant difference in myocardial tissue K⁺ among the four groups. There was a significant difference in the arrhythmic dose of epinephrine among the four groups (P < 0.05). Acute hypokalemia was more prone to dysrhythmias than chronic hypokalemia. However, compared with control, acute and chronic hypokalemia groups were resistant to dysrhythmias during anesthesia. The resistance to dysrhythmias is probably based on compensatory mechanisms. The heart seems more resistant to K⁺ changes than skeletal muscle. This resistance is associated with compensation by the cardiac muscle sodium pump in the face of K⁺ depletion. Hypokalemia per se did not increase the incidence of dysrhythmias under halothane anesthesia in rats.     

Ca2+-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers

Summary This study evaluates the use of the planar lipid bilayer as a functional assay of Ca²⁺-activated K⁺ channel activity for use in purification of the channel protein. Ca²⁺-activated K⁺ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K⁺-selective (p_Cl/p_K=0.046; P_Na/P_K=0.027). Channels incorporated into the bilayer with two orientations. In 65 % of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca²⁺ concentration of about 15 mol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca²⁺ activated the channel. These results resemble those found for Ca²⁺-activated K⁺ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.     

Permeability of single muscle capillaries to potassium ions

A frog muscle preparation suitable for capillary micropuncture is described. The K⁺ permeability of single muscle capillaries was measured using the methods developed for frog mesenteric capillaries (C. Crone, J. Frøkjaer-Jensen, J. J. Friedman, and O. Christensen (1978), J. Gen. Physiol.71, 195–220). The K⁺ permeability of arterial capillaries was 8.6 × 10^?5 cm/sec (SD = 1.8; n = 7). The initial K⁺ permeability of venous capillaries was 13.1 × 10^?5 cm/sec (SD = 4.3; n = 6). In venous capillaries—unlike in arterial capillaries—a gradual increase in permeability associated with endothelial gap formation was observed in repeated measurements on the same capillary segment. This inflammatory response to exposure, micromanipulation, or perfusion could be partially blocked by pretreatment with promethazine. The study shows that frog muscle capillaries differ markedly from frog mesenteric capillaries, being 5–10 times less permeable to potassium ions although both belong to the category of continuous capillaries. The results demonstrate that whole-organ and single-capillary techniques for studying muscle capillary permeability yield values which comply within a factor of about 2.