The local differentiation of myelinated axons at nodes of Ranvier

Efficient and rapid propagation of action potentials in myelinated axons depends on the molecular specialization of the nodes of Ranvier. The nodal region is organized into several distinct domains, each of which contains a unique set of ion channels, cell-adhesion molecules and cytoplasmic adaptor proteins. Voltage-gated Na+ channels - which are concentrated at the nodes - are separated from K+ channels - which are clustered at the juxtaparanodal region - by a specialized axoglial contact that is formed between the axon and the myelinating cell at the paranodes. This local differentiation of myelinated axons is tightly regulated by oligodendrocytes and myelinating Schwann cells, and is achieved through complex mechanisms that are used by another specialized cell-cell contact - the synapse.     

Kinetic methods of reverse mechanism functions of receptor domain Na+- and K+-channels]

Complete kinetic diagrams of the gate mechanisms of Na(+)- and K(+)-channels of receptor domains were examined. Analysis of various states of gate mechanisms of these channels allow to reveal the fundamental characteristics of Na(+)- and K(+)-channels of receptor domains which can underlie the molecular mechanisms of a number of processes dealing with the functioning of receptor domains. Taking into account these characteristics of Na(+)- and K(+)-channels of receptor domains, the molecular mechanisms of generation and spreading of the action potential and the molecular mechanisms of synaptic transmission, in chemical, and electrical synapses, was proposed. A possible role of receptor domains in the processes of cell differentiation, proliferation and carcinogenesis are also discussed.     

K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve

The localization of Shaker-type K⁺ channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic β-subunit Kvβ2 were colocalized in juxtaparanodal zones. During development, clustering of K⁺ channels lagged behind that for nodal Na⁺ channels by about 5 days. In contrast to the PNS, K⁺ channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K⁺ channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na⁺ channel clusters, and little or no detectable clustering of K⁺ channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K⁺ channel clustering and localization.     

Clockwise domain arrangement of the sodium channel revealed by (mu)-conotoxin (GIIIA) docking orientation

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.     

Neurofascin assembles a specialized extracellular matrix at the axon initial segment

Action potential initiation and propagation requires clustered Na(+) (voltage-gated Na(+) [Nav]) channels at axon initial segments (AIS) and nodes of Ranvier. In addition to ion channels, these domains are characterized by cell adhesion molecules (CAMs; neurofascin-186 [NF-186] and neuron glia-related CAM [NrCAM]), cytoskeletal proteins (ankyrinG and betaIV spectrin), and the extracellular chondroitin-sulfate proteoglycan brevican. Schwann cells initiate peripheral nervous system node formation by clustering NF-186, which then recruits ankyrinG and Nav channels. However, AIS assembly of this protein complex does not require glial contact. To determine the AIS assembly mechanism, we silenced expression of AIS proteins by RNA interference. AnkyrinG knockdown prevented AIS localization of all other AIS proteins. Loss of NF-186, NrCAM, Nav channels, or betaIV spectrin did not affect other neuronal AIS proteins. However, loss of NF-186 blocked assembly of the brevican-based AIS extracellular matrix, and NF-186 overexpression caused somatodendritic brevican clustering. Thus, NF-186 assembles and links the specialized brevican-containing AIS extracellular matrix to the intracellular cytoskeleton.     

Molecular domains of myelinated axons

Myelinated axons are organized into specific domains as the result of interactions with glial cells. Recently, distinct protein complexes of cell adhesion molecules, Na(+) channels and ankyrin G at the nodes, Caspr and contactin in the paranodes, and K(+) channels and Caspr2 in the juxtaparanodal region have been identified, and new insights into the role of the paranodal junctions in the organization of these domains have emerged.     

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Essential role of oligodendrocytes in the formation and maintenance of central nervous system nodal regions.

The membrane of myelinated axons is divided into functionally distinct domains characterized by the enrichment of specific proteins. The mechanisms responsible for this organization have not been fully identified. To further address the role of oligodendrocytes in the functional segmentation of the axolemma in vivo, the distribution of nodal (Na(+) channels, ankyrin G), paranodal (paranodin/contactin-associated-protein) and juxtaparanodal (Kv1.1 K(+) channels) axonal markers, was studied in the brain of MBP-TK and jimpy mice. In MBP-TK transgenic mice, oligodendrocyte ablation was selectively induced by FIAU treatment before and during the onset of myelination. In jimpy mice, oligodendrocytes degenerate spontaneously within the first postnatal weeks after the onset of myelination. Interestingly, in MBP-TK mice treated for 1-20 days with FIAU, despite the ablation of more than 95% of oligodendrocytes, the protein levels of all tested nodal markers was unaltered. Nevertheless, these proteins failed to cluster in the nodal regions. By contrast, in jimpy mice, despite a diffused localization of paranodin, the formation of nodal clusters of Na(+) channels and ankyrin G was observed. Furthermore, K(+) channels clusters were transiently visible, but were in direct contact with nodal markers. These results demonstrate that the organization of functional domains in myelinated axons is oligodendrocyte dependent. They also show that the presence of these cells is a requirement for the maintenance of nodal and paranodal regions.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.     

Second transmembrane domains of ENaC subunits contribute to ion permeation and selectivity

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.     

A family of calcium-dependent potassium channels from rat brain

By incorporating rat brain plasma membrane vesicles into planar lipid bilayers, we have found and characterized four types of Ca2(+)-activated K+ channels. The unitary conductances of these channels are 242 +/- 14 pS, 236 +/- 16 pS, 135 +/- 10 pS, and 76 +/- 6 pS in symmetrical 150 mM KCI buffers. These channels share a number of properties. They are all activated by depolarizing voltages, activated by micromolar concentrations of internal Ca2+ with a Hill coefficient for Ca2+ activation of between 2 and 3, noninactivating under our assay conditions, blocked by low millimolar concentrations of TEA from the outside, apamin-insensitive, and very selective for K+ over Na+ and Cl-. Three of the four channels are also blocked by nanomolar concentrations of charybdotoxin. One of the high conductance Ca2(+)-activated K+ channels is novel in that it is not blocked by charybdotoxin and exhibits gating kinetics highlighted by long closed times and long open times. This family of closely related Ca2(+)-activated K+ channels may share structural domains underlying particular functions.     

Towards a structural view of gating in potassium channels

Voltage-activated cation channels have pores that are selective for K(+), Na(+) or Ca(2+). Neurons use these channels to generate and propagate action potentials, release neurotransmitters at synaptic terminals and integrate incoming signals in dendrites. Recent X-ray and electron microscopy studies of an archaebacterial voltage-activated K(+) (Kv) channel have provided the first atomic resolution images of the voltage-sensing domains in Kv channels. Although these structures are consistent with previous biophysical analyses of eukaryotic channels, they also contain surprises, which have provoked new ideas about the structure and movements of these proteins during gating. This review summarizes our current understanding of these intriguing membrane proteins and highlights the open questions.     

K+/Na+ selectivity in K channels and valinomycin: over-coordination versus cavity-size constraints

Potassium channels and valinomycin molecules share the exquisite ability to select K(+) over Na(+). Highly selective K channels maintain a special local environment around their binding sites devoid of competing hydrogen bond donor groups, which enables spontaneous transfer of K(+) from states of low coordinations in water into states of over-coordination by eight carbonyl ligands. In such a phase-activated state, electrostatic interactions from these 8-fold binding sites, constrained to maintain high coordinations, result in K(+)/Na(+) selectivity with no need for a specific cavity size. Under such conditions, however, direct coordination from five or six carbonyl ligands does not result in selectivity. Yet, valinomycin molecules achieve selectivity by providing only six carbonyl ligands. Does valinomycin use additional coordinating ligands from the solvent or does it have special structural features not present in K channels? Quantum chemical investigations undertaken here demonstrate that valinomycin selectivity is due to cavity size constraints that physically prevent it from collapsing onto the smaller sodium ion. Valinomycin enforces these constraints by using a combination of intramolecular hydrogen bonds and other structural features, including its specific ring size and the spacing between its connected ligands. Results of these investigations provide a consistent explanation for the experimental data available for the ion-complexation properties of valinomycin in solvents of varying polarity. Together, investigations of these two systems reveal how nature, despite being popular for its parsimony in recycling functional motifs, can use different combinations of phase, coordination number, cavity size, and rigidity (constraints) to achieve K(+)/Na(+) selectivity.     

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution: recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis

Unidirectional (22)Na, Li(+) and Rb(+) fluxes and net fluxes of Na(+) and K(+) were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 microM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na(+) and K(+) efflux and influx, i.e. fluxes via the Na(+),K(+)-ATPase pump, Na(+) channels, K(+) channels, Na/Na exchanger and Na-Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na(+) and K(+) fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na-Cl symport and integral Na(+) channel permeability.     

A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K(+) influx is neither inhibited nor stimulated by Na(+). Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na(+) transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance.     

Na(+) current through KATP channels: consequences for Na(+) and K(+) fluxes during early myocardial ischemia

During early myocardial ischemia, the myocytes are loaded with Na(+), which in turn leads to Ca(2+) overload and cell death. The pathway of the Na(+) influx has not been fully elucidated. The aim of the study was to quantify the Na(+) inward current through sarcolemmal KATP channels (IKATP,Na) in anoxic isolated cardiomyocytes at the actual reversal potential (Vrev) and to estimate the contribution of this current to the Na(+) influx in the ischemic myocardium. IKATP,Na was determined in excised single channel patches of mouse ventricular myocytes and macropatches of Xenopus laevis oocytes expressing SUR2A/Kir6.2 channels. In the presence of K+ ions, the respective permeability ratios for Na(+) to K(+) ions, PNa/PK, were close to 0.01. Only in the presence of Na(+) ions on both sides of the membrane was IKATP,Na similarly large to that calculated from the permeability ratio PNa/PK, indicative of a Na(+) influx that is largely independent of the K+ efflux at Vrev. With the use of a peak KATP channel conductance in anoxic cardiomyocytes of 410 nS, model simulations for a myocyte within the ischemic myocardium showed that the amplitude of the Na(+) influx and K(+) efflux is even larger than the respective fluxes by the Na(+) - K(+) pump and all other background fluxes. These results suggest that during early ischemia the Na(+) influx through KATP channels essentially contributes to the total Na+ influx and that it also balances the K(+) efflux through KATP channels.     

The interaction of Na(+) and K(+) in the pore of cyclic nucleotide-gated channels

The permeability ratio between K(+) and Na(+) ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K(+) or Na(+). Therefore, K(+) and Na(+) ions are thought to permeate with identical properties. In the alpha-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K(+) and Na(+) ions. Indeed K(+), Rb(+) and Cs(+) ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na(+) ions. Moreover in mutant P365T the presence of K(+) in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na(+) when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na(+) is replaced with K(+). As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K(+) ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K(+) ion is driven out. Potentiation occurs because K(+) ions slow down the rate constant K(off) controlling channel closure. These results indicate that K(+) and Na(+) ions do not permeate through CNG channels in the same way and that K(+) ions influence the channel gating.     

Second transmembrane domain modulates epithelial sodium channel gating in response to shear stress

Na(+) absorption and K(+) secretion in the distal segments of the nephron are modulated by the tubular flow rate. Epithelial Na(+) channels (ENaC), composed of α-, β-, and γ-subunits respond to laminar shear stress (LSS) with an increase in open probability. Higher vertebrates express a δ-ENaC subunit that is functionally related to the α-subunit, while sharing only 35% of sequence identity. We investigated the response of δβγ channels to LSS. Both the time course and magnitude of activation of δβγ channels by LSS were remarkably different from those of αβγ channels. ENaC subunits have similar topology, with an extracellular region connected by two transmembrane domains with intracellular N and C termini. To identify the specific domains that are responsible for the differences in the response to flow of αβγ and δβγ channels, we generated a series of α-δ chimeras and site-specific α-subunit mutants and examined parameters of activation by LSS. We found that specific sites in the region encompassing and just preceding the second transmembrane domain were responsible for the differences in the magnitude and time course of channel activation by LSS.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.