Scorpion neurotoxin - a presynaptic toxin which affects both Na+ and K+ channels in axons.

A neurotoxin from the venom of the scorpion, $\text{Androctonus australis Hector}$, affects the closing of the Na⁺ channel and the opening of the K⁺ channel in giant axons of crayfish and lobster nerves. It blocks both Na⁺ and K⁺ conductances in $\text{Sepia}$ giant axons. Dose-response curves are markedly cooperative with all types of axons. Apparent dissociation constants for the receptor-toxin complexes are 0.25 μM, 0.7 μM and 2–4 μM for the crayfish, lobster and $\text{Sepia}$ axons, respectively. This toxin will be probably a useful tool for biochemical investigation of Na⁺ and K⁺ channels.     

Prolactin, an activator of epithelial Na+ channel, inhibits basolateral K+ channels in adult tree frog skin

Adult amphibian skin actively transports Na+ from its apical to basolateral side while in turn, K+ is recycled through Na+, K+-ATPase and K+ channels located in the basolateral membrane. We previously found that PRL stimulates Na+ transport in the skin of the adult tree frog (Hyla arborea japonica) via an increase in the open-channel density of the epithelial Na+ channel (ENaC). If PRL also activates basolateral K+ channels, this activation would help to stimulate Na+ transport, too. Whether PRL does indeed stimulate basolateral K+ channels in the adult tree frog was examined by measuring the short-circuit current across nystatin-treated skin. Both tolbutamide, a K(ATP) channel blocker, and tetrapentylammonium (TPA), a KCa channel blocker, blocked the current, the effect of TPA being more powerful than that of tolbutamide. Contrary to expectation, PRL inhibited the basolateral K+ channels in this skin. In the presence of basolateral amiloride, PRL still inhibited the basolateral K+ current, suggesting that the (Na+)-H+ exchanger located in the basolateral membrane does not mediate the inhibitory effect of PRL on the basolateral K+ channels in Hyla.     

Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.     

Fluctuation analysis of Na+ channels modified by batrachotoxin in myelinated nerve

(1) Single myelinated nerve fibers of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na+ currents and Na+-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses the fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na+-current fluctuations was fitted by the sum of a 1/f component and a Lorentzian function. The time constant tau c = 1/(2 pi fc) obtained from the corner frequency fc of the Lorentzian function approximately agreed with the activation time constant tau m of the macroscopic currents. (3) The conductance gamma of a single Na+ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na+ current. At the resting potential V = 0 we obtained gamma - 1.6 pS, higher gamma-values of 3.2 and 3.45 pS were found at V = --8 and --16 mV, respectively. (4) The conductance of a modified Na+ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na+ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an 'all-or-none' manner on Na+ channels and creates a distinct population of modified channels.     

Gating at the selectivity filter of ion channels that conduct Na+ and K+ ions

The NaK channel is a cation selective channel with similar permeability for K⁺ and Na⁺. The available crystallographic structure of wild-type (WT) NaK is usually associated with a conductive state of the channel. Here, potential of mean force for complete conduction events of Na⁺ and K⁺ ions through NaK show that: i), large energy barriers prevent the passage of ions through the WT NaK structure, ii), the barriers are correlated to the presence of a hydrogen bond between Asp-66 and Asn-68, and iii), the structure of NaK mutated to mimic cyclic nucleotide-gated channels conducts Na⁺ and K⁺. These results support the hypothesis that the filter of cation selective channels can adopt at least two different structures: a conductive one, represented by the x-ray structures of the NaK-CNG chimeras, and a closed one, represented by the x-ray structures of the WT NaK.     

Na+ and K+ channels: history and structure

In this perspective, we discuss the physiological roles of Na and K channels, emphasizing the importance of the K channel for cellular homeostasis in animal cells and of Na and K channels for cellular signaling. We consider the structural basis of Na and K channel gating in light of recent structural and electrophysiological findings.     

Properties of single Na+ channels in cut-open Myxicola giant axons

Time- and voltage-dependent behavior of the Na+ conductance in dialyzed intact Myxicola axons was compared with cut-open axons subjected to loose-patch clamp of the interior and to axons where Gigaseals were formed after brief enzyme digestion. Voltage and time dependence of activation, inactivation, and reactivation were identical in whole-axons and loose-patch preparations. Single channels observed in patch-clamp axons had a conductance of 18.3 +/- 2.3 pS and a mean open time of 0.84 +/- 0.12 ms. The time-dependence of Na+ currents found by averaging patch-clamp records was similar to intact axons, as was the voltage dependence of activation. Steady-state inactivation in patch-clamped axons was shifted by an average of 15 mV from that seen in loose-patch or intact axons. Substitution of D2O for H2O decreased single channel conductance by 24 +/- 6% in patch-clamped axons compared with 28 +/- 4% in intact axons, slowed inactivation by 58 +/- 8% compared with 49 +/- 6%, and increased mean open time by 52 +/- 7%. The results confirm observations on macroscopic channel behavior in Myxicola and resemble that seen in other excitable tissues.     

Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Here, we describe the identification of Caspr2, a mammalian homolog of Drosophila Neurexin IV (Nrx-IV), and show that this neurexin-like protein and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. We further show that Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains a putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains.     

Single-file diffusion through K+ channels in frog skin epithelium

Summary The ratio between the unidirectional fluxes of K⁺ across the frog skin with K-permeable outer membranes was determined in the absence of Na⁺ in the apical solutions. The experiments were performed under presteady-state conditions to be able to separate the flux ratio for K⁺ through the cells from contributions to the fluxes through extracellular leaks. The cellular flux ratio deviated strongly from the value calculated from the flux ratio for electrodiffusion. The experiments can be explained if the passive K transport through the epithelial cells proceeds through specific channels by single-file diffusion with a flux ratio exponent of about 2.5.     

Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.     

Removal of Na+ channels in squid giant axons by perfusion with trypsin

The irreversible effects of the proteolytic enzyme trypsin on ionic and gating currents of voltage-clamped squid axon membranes have been studied. At physiological pH, internal perfusion of the fibre with trypsin was found to be very effective in removing Na+ channels leaving the potassium system almost unaltered. At T = 13 degrees C the rates of channel-cleavage averaged 1/10 min-1 for the Na+ and 1/128 min-1 for the K+ channel, respectively. As estimated by the decrement of peak sodium conductance, the rate of loss of Na+ channels correlates well with the rate of decrease of the total charge associated with the ON component of gating currents, indicating that trypsin probably interacts with an essential proteic portion of the channel whose removal might prevent both the displacement of gating charges and the subsequent opening of the channel. Intracellular pH remarkably influences the action of the enzyme. A plot of the pH-dependence of the rate of cleavage of Na+ channels suggests the involvement of a positively charged group (either lysine or arginine) in the substrate region of the trypsin catalytic reaction.     

Clustering and activity tuning of Kv1 channels in myelinated hippocampal axons

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.     

Activity coefficients of intracellular Na+ and K+ during development of frog oocytes

Summary The chemical activities, (a), of Na⁺ and K⁺ were determined in large mature and in small immature frog oocytes, using open-tipped micropipettes and ionselective microelectrodes. The average chemical concentrations,c, of Na⁺ and K⁺ were determined by spectrophotometry and by electron probe X-ray microanalysis. The apparent activity coefficient (^app) was calculated for each ion as the ratio,a/c.With development, (a _Na/a _K) decreased four to fivefold and (c _Na/c _K) increased six to sevenfold. In the large mature oocytes, _Na ^app was measured to be 0.08±0.02 and _K ^app lay within the range 1.15±0.03 to 1.29±0.04, constituting the smallest value for Na⁺ and largest value for K⁺, respectively, thus far reported. This intracellular value of _K ^app was substantially greater than the activity coefficient of K⁺ in the external medium (0.76). The data suggest that the inequality of _Na ^app and _K ^app in this and probably other cells reflects the development of subcellular compartmentalization of ions. Possible intracellular sites of ionic compartmentalization are considered.     

Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration

Epithelial cell migration plays an important role in gastrointestinal mucosal repair. We previously reported that multiple functional ion channels, including a Ba²⁺-sensitive K⁺ inward rectifier K_ir1.2, 4-aminopyridine (4-AP)-sensitive voltage-gated K⁺ channels K_v1.1, K_v1.6 and K_v2.1, and a nifedipine-sensitive, tetrodotoxin (TTX)-insensitive voltage-gated Na⁺ channel Na_v1.5 were expressed in a non-transformed rat gastric epithelial cell line (RGM-1). In the present study, we further investigated whether these ion channels are involved in the modulation of gastric epithelial cell migration. Cell migration was determined by monolayer wound healing assay. Results showed that blockade of K_v with 4-AP or Na_v1.5 with nifedipine inhibited RGM-1 cell migration in the absence or presence of epidermal growth factor (EGF), which effectively stimulated RGM-1 cell migration. Moreover, high concentration of TTX mimicked the action of nifedipine, suggesting that the action of nifedipine was mediated through specific blockade of Na_v1.5. In contrast, inhibition of K_ir1.2 with Ba²⁺, either in basal or EGF-stimulated condition, had no effect on RGM-1 cell migration. In conclusion, the present study demonstrates for the first time that voltage-gated K⁺ and Na⁺ channels are involved in the modulation of gastric epithelial cell migration.     

Rapid and slow gating of veratridine-modified sodium channels in frog myelinated nerve

The properties of voltage-dependent Na channels modified by veratridine (VTD) were studied in voltage-clamped nodes of Ranvier of the frog Rana pipiens. Two modes of gating of VTD-modified channels are described. The first, occurring on a time scale of milliseconds, is shown to be the transition of channels between a modified resting state and a modified open state. There are important qualitative and quantitative differences of this gating process in nerve compared with that in muscle (Leibowitz et al., 1986). A second gating process occurring on a time scale of seconds, was originally described as a modified activation process (Ulbricht, 1969). This process is further analyzed here, and a model is presented in which the slow process represents the gating of VTD-modified channels between open and inactivated states. An expanded model is a step in the direction of unifying the known rapid and slow physiologic processes of Na channels modified by VTD and related alkaloid neurotoxins.