Interaction between fast and slow inactivation in Skm1 sodium channels.

Rat skeletal muscle (Skm1) sodium channel alpha and beta 1 subunits were coexpressed in Xenopus oocytes, and resulting sodium currents were recorded from on-cell macropatches. First, the kinetics and steady-state probability of both fast and slow inactivation in Skm1 wild type (WT) sodium channels were characterized. Next, we confirmed that mutation of IFM to QQQ (IFM1303QQQ) in the DIII-IV 'inactivation loop' completely removed fast inactivation at all voltages. This mutation was then used to characterize Skm1 slow inactivation without the presence of fast inactivation. The major findings of this paper are as follows: 1) Even with complete removal of fast inactivation by the IFM1303QQQ mutation, slow inactivation remains intact. 2) In WT channels, approximately 20% of channels fail to slow-inactivate after fast-inactivating, even at very positive potentials. 3) Selective removal of fast inactivation by IFM1303QQQ allows slow inactivation to occur more quickly and completely than in WT. We conclude that fast inactivation reduces the probability of subsequent slow inactivation.     

Slow inactivation in voltage-gated sodium channels

Slow inactivation in voltage-gated sodium channels is a biophysical process that governs the availability of sodium channels over extended periods of time. Slow inactivation, therefore, plays an important role in controlling membrane excitability, firing properties, and spike frequency adaptation. Defective slow inactivation is associated with several diseases of cell excitability, such as hyperkalemic periodic paralysis, myotonia, idiopathic ventricular fibrillation and long-QT syndrome. These associations underscore the physiological importance of this phenomenon. Nevertheless, our understanding of the molecular substrates for slow inactivation is still fragmentary. This review covers the current state of knowledge concerning the molecular underpinnings of slow inactivation, and its relationship with other biophysical processes of voltage-gated sodium channels.     

Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

The human ether-á-go-go–related gene (hERG) K⁺ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, I_Kr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of I_Kr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q₁ and Q₂, with V_1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q₂ charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q₁ and Q₂, decreasing to 4.3 ms for Q₂ at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q₂ movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V_1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q₁ and Q₂ charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.     

Implication of the C-terminal region of the alpha-subunit of voltage-gated sodium channels in fast inactivation

The α-subunit of both the human heart (hH1) and human skeletal muscle (hSkM1) sodium channels were expressed in a mammalian expression system. The channels displayed slow (hH1) and fast (hSkM1) current decay kinetics similar to those seen in native tissues. Hence, the aim of this study was to identify the region on the α-subunit involved in the differences of these current-decay kinetics. A series of hH1/hSkM1 chimeric sodium channels were constructed with the focus on the C-terminal region. Sodium currents of chimeric channels were recorded using the patch-clamp technique in whole-cell configuration. Chimeras where the C-terminal region had been exchanged between hH1 and hSkM1 revealed that this region contains the elements that cause differences in current decay kinetics between these sodium channel isoforms. Other biophysical characteristics (steady-state activation and inactivation and recovery from inactivation) were similar to the phenotype of the parent channel. This indicates that the C-terminus is exclusively implicated in the differences of current decay kinetics. Several other chimeras were constructed to identify a specific region of the C-terminus causing this difference. Our results showed that the first 100-amino-acid stretch of the C-terminal region contains constituents that could cause the differences in current decay between the heart and skeletal muscle sodium channels. This study has uncovered a direct relationship between the C-terminal region and the current-decay of sodium channels. These findings support the premise that a novel regulatory component exists for fast inactivation of voltage-gated sodium channels. Received: 1 March 2001/Revised: 18 May 2001     

Open- and closed-state fast inactivation in sodium channels: differential effects of a site-3 anemone toxin

The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.     

Alpha-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels

Alpha-scorpion toxins bind in a voltage-dependent way to site 3 of the sodium channels, which is partially formed by the loop connecting S3 and S4 segments of domain IV, slowing down fast inactivation. We have used Ts3, an alpha-scorpion toxin from the Brazilian scorpion Tityus serrulatus, to analyze the effects of this family of toxins on the muscle sodium channels expressed in Xenopus oocytes. In the presence of Ts3 the total gating charge was reduced by 30% compared with control conditions. Ts3 accelerated the gating current kinetics, decreasing the contribution of the slow component to the ON gating current decay, indicating that S4-DIV was specifically inhibited by the toxin. In addition, Ts3 accelerated and decreased the fraction of charge in the slow component of the OFF gating current decay, which reflects an acceleration in the recovery from the fast inactivation. Site-specific fluorescence measurements indicate that Ts3 binding to the voltage-gated sodium channel eliminates one of the components of the fluorescent signal from S4-DIV. We also measured the fluorescent signals produced by the movement of the first three voltage sensors to test whether the bound Ts3 affects the movement of the other voltage sensors. While the fluorescence-voltage (F-V) relationship of domain II was only slightly affected and the F-V of domain III remained unaffected in the presence of Ts3, the toxin significantly shifted the F-V of domain I to more positive potentials, which agrees with previous studies showing a strong coupling between domains I and IV. These results are consistent with the proposed model, in which Ts3 specifically impairs the fraction of the movement of the S4-DIV that allows fast inactivation to occur at normal rates.     

Impaired slow inactivation in mutant sodium channels.

Hyperkalemic periodic paralysis (HyperPP) is a disorder in which current through Na+ channels causes a prolonged depolarization of skeletal muscle fibers, resulting in membrane inexcitability and muscle paralysis. Although HyperPP mutations can enhance persistent sodium currents, unaltered slow inactivation would effectively eliminate any sustained currents through the mutant channels. We now report that rat skeletal muscle channels containing the mutation T698M, which corresponds to the human T704M HyperPP mutation, recover very quickly from prolonged depolarizations. Even after holding at -20 mV for 20 min, approximately 25% of the maximal sodium current is available subsequent to a 10-ms hyperpolarization (-100 mV). Under the same conditions, recovery is less than 3% in wild-type channels and in the F1304Q mutant, which has impaired fast inactivation. This effect of the T698M mutation on slow inactivation, in combination with its effects on activation, is expected to result in persistent currents such as that seen in HyperPP muscle.     

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.     

A molecular link between activation and inactivation of sodium channels

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.     

Human movement is both diffusive and directed

Understanding the influence of the built environment on human movement requires quantifying spatial structure in a general sense. Because of the difficulty of this task, studies of movement dynamics often ignore spatial heterogeneity and treat movement through journey lengths or distances alone. This study analyses public bicycle data from central London to reveal that, although journey distances, directions, and frequencies of occurrence are spatially variable, their relative spatial patterns remain largely constant, suggesting the influence of a fixed spatial template. A method is presented to describe this underlying space in terms of the relative orientation of movements toward, away from, and around locations of geographical or cultural significance. This produces two fields: one of convergence and one of divergence, which are able to accurately reconstruct the observed spatial variations in movement. These two fields also reveal categorical distinctions between shorter journeys merely serving diffusion away from significant locations, and longer journeys intentionally serving transport between spatially distinct centres of collective importance. Collective patterns of human movement are thus revealed to arise from a combination of both diffusive and directed movement, with aggregate statistics such as mean travel distances primarily determined by relative numbers of these two kinds of journeys.     

RepC is rate limiting for pT181 plasmid replication

The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.     

A conserved glutamate is important for slow inactivation in K+ channels

Voltage-gated ion channels undergo slow inactivation during prolonged depolarizations. We investigated the role of a conserved glutamate at the extracellular end of segment 5 (S5) in slow inactivation by mutating it to a cysteine (E418C in Shaker). We could lock the channel in two different conformations by disulfide-linking 418C to two different cysteines, introduced in the Pore-S6 (P-S6) loop. Our results suggest that E418 is normally stabilizing the open conformation of the slow inactivation gate by forming hydrogen bonds with the P-S6 loop. Breaking these bonds allows the P-S6 loop to rotate, which closes the slow inactivation gate. Our results also suggest a mechanism of how the movement of the voltage sensor can induce slow inactivation by destabilizing these bonds.     

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons

Summary Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0–8°C. The time course of the birefringence changes share many properties of the gating (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30mm colchicine; neither are altered by changes on sodium concentrations or 300nm tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation.The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.     

Effects of chloramine-T on activation and inactivation of sodium channels in neuroblastoma cells

The effects of chloramine-T, a reagent specific to methionine residues, on sodium channel gating mechanisms was investigated in neuroblastoma cell membrane. Treating the membrane with chloramine was found to retard inactivation kinetics and considerably reduce the slope of the inactivation curve, while pushing the activation curve toward hyperpolarization ranges without changing the slope of the central portion perceptibly. Effective activation charge, as determined from the limiting logarithmic slope of activation, was reduced by a factor of 1.17. Possible reasons for the changes observed in sodium channel gating mechanisms are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 789–795, November–December, 1987.     

Activation and inactivation of batrachotoxin-modified sodium channels in the frog nerve fiber membrane

Currents through batrachotoxin (BTX)-modified sodium channels were measured under voltage clamp conditions on the Ranvier node membrane. Potential-dependence of the fraction of activated BTX-modified channels was determined on the basis of data showing nonlinearity of the momentary current-voltage characteristic curve in the region of high negative potentials. BTX induces a shift of the sodium channel activation curve toward negative potentials on average by 67 mV, but does not, under these circumstances, alter the potential-sensitivity of their activation mechanism. The results of experiments with preliminary depolarization, of varied amplitude and duration, showed that BTX-modified sodium channels are capable of partial inactivation. The high level of steady-state conduction of the modified channels is evidently due to the fact that as a result of modification by BTX the open state of the channel becomes energetically more advantageous than the inactivated state. It is concluded that the action of BTX on inactivation differs in principle from the action of pronase.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 1, pp. 18–26. January–February, 1984.     

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.     

Domain I of ribosomal protein L1 is sufficient for specific RNA binding

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.     

Some kinetic and steady-state properties of sodium channels after removal of inactivation

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sympathetic innervation of lymphoreticular organs is rate limiting for prion neuroinvasion

Transmissible spongiform encephalopathies are commonly propagated by extracerebral inoculation of the infectious agent. Indirect evidence suggests that entry into the central nervous system occurs via the peripheral nervous system. Here we have investigated the role of the sympathetic nervous system in prion neuroinvasion. Following intraperitoneal prion inoculation, chemical or immunological sympathectomy delayed or prevented scrapie. Prion titers in spinal cords were drastically reduced at early time points after inoculation. Instead, keratin 14-NGF transgenic mice, whose lymphoid organs are hyperinnervated by sympathetic nerves, showed reduction in scrapie incubation time and, unexpectedly, much higher titers of prion infectivity in spleens. We conclude that sympathetic innervation of lymphoid organs is rate limiting for prion neuroinvasion and that splenic sympathetic nerves may act as extracerebral prion reservoirs.