Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Structure,development and function of the branchial sieve of the common bream,Abramis brama,white bream,Blicca bjoerkna and roach,Rutilus rutilus

Synopsis The filter feeding organ of cyprinid fishes is the branchial sieve, which consists of a mesh formed by gill rakers and tiny channels on the gill arches. In order to establish its possible role during growth we measured the following morphological gill raker parameters over a range of sizes in three cyprinid fishes, bream, white bream and roach: inter raker distance, bony raker length, raker width, cushion length and channel width. At any given standard length common bream has the largest inter raker distance, roach the lowest and white bream is intermediate. In the comb model of filter feeding the inter raker distance is considered to be a direct measure of the mesh size and retention ability (= minimal size of prey that can be retained) of a filter. For the three species under study there is a conflict between the comb model and experimental data on particle retention. Lammens et al. (1987) found that common bream has a large retention ability whereas roach and white bream have a much smaller one. A new model, the channel model (Hoogenboezem et al. 1991) has been developed for common bream; in this model the lateral gill rakers can regulate the mesh size of the medial channels on the other side of the gill slit. The present data indicate that this model is not appropriate for white bream and roach. At any given standard length white bream and roach only reach 70% of the raker length of common bream, which means that in this model the gill slits should to be very narrow during filter feeding. The gill rakers consist of a bony raker and a fleshy cushion. The bony rakers have a rather long needle-like part outside the cushion in bream, but not in white bream and roach which have blunt gill rakers. Blunt gill rakers are not suited to reduce the diameter of the medial channels. The comb model seems more appropriate for white bream and roach, but doubts about the validity of this simple model remain. The sum of the areas of the medial channels is an approximation of the area through which water flows in the filter. This channel area therefore gives an impression of the capacity or flow rate of the filter. With this capacity estimation and an estimation of energy consumption we calculated an energy ratio of filter feeding. The energy ratio decreases with increasing standard length with an exponent close to the expected exponent of -0.40. The energy ratio is highest in bream, intermediate in white bream and lowest in roach.     

Effect of initial size on subsequent growth and carcass characteristics of divergently selected channel catfish

Summary Two experiments were conducted simultaneously to determine (1) if fast-growing fingerlings of channel catfish, Ictalurus punctatus, could be identified by simple visual selection of body size and (2) if initial size advantages influenced subsequent growth and carcass traits of divergently selected channel catfish. Exp. 1 included large (L), medium (M), and small (S) fingerling sizes from each of the control (C), selected upward (+) and selected downward (–) lines for body weight. Exp. 2 included all fmgerlings of the same size (25±5 g) from the 3 lines. Catfish from the L size-class, within each full-sib family in each line, were consistently heavier and longer than M and S size-classes throughout the 53-week experimental period. Fingerlings from the M size-class were also superior in growth to those from the S size-class. Catfish from the + line exceeded those from the C and –lines in body weight and total length at the conclusion of Exp. 1 but not in Exp. 2. This was attributed to the selection of equal size fmgerlings in Exp. 2 which may have excluded fingerlings with the best growth potential from the + body weight line. Results of the two experiments combined indicated that one generation of divergent selection has created genetic differences among lines of channel catfish.Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Station     

Ionic channels through the axon membrane (a review)

Ionic channels are discrete sites at which the passive movement of ions takes place during nervous excitation. Three types of channels are distinguished. 1. Leakage channels that are permanently open to various cations. 2. Na channels that open promptly on depolarization but slowly close again (inactivate) on sustained depolarization and that are predominantly permeable to Na⁺ ions. 3. K channels that on depolarization open after some delay but stay open and that are mainly passed by K⁺ ions. The selectivity sequence of the Na channels of the squid axon (or frog nerve) is as follows: Na⁺ ≈ Li⁺>(T1+)>NH⁺ ₄?K⁺> Rb⁺, Cs⁺; that of K channels is: (T1+)>K⁺>Rb⁺>NH⁺ ₄?Na⁺, Cs⁺, Na channels are selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX), K channels by tetraethylammonium ions (TEA). Either channel type is reversibly blocked when one drug molecule binds to one site per channel, the equilibrium dissociation constant of these reactions being about 3×10^?9 MTTX (or STX) and 4×10^?4 M TEA, respectively. Because of their specificity and high affinity, TTX and STX are used to “titrate” the Na channels whose density appears to be of the order of 100/Μm². The “gates” of the channels operate as a function of potential and time but independent of the permeating ion species. Drugs (e.g. veratridine) and enzymes (e.g. pronase, applied intraaxonally) cause profound changes in the gating function of the Na channels without influencing their selectivity. This points to separate structures for gating and ion discrimination. The latter is thought to be, in part, brought about by a “selectivity filter” of which detailed structural ideas exist. Recent experiments suggest that the gates of the Na channels are controlled by charged particles moving within the membrane under the influence of the electrical field.     

Identification of a cationic channel in synaptosomal membranes

Synaptosomal membranes were fused with liposomes using the hydration technique to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters. Offprint requests to: H. Breer     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Ion channel regulation by calmodulin binding

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca²⁺-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca²⁺-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.