A tetrodotoxin-resistant voltage-gated Na ϩ current (TTX-R I Na ) appears to be the current primarily responsible for action potential generation in the cell body and terminals of nociceptive afferents. Although other voltage-gated Na ϩ currents are modulated by the activation of protein kinase C (PKC), protein kinase A (PKA), or both, the second messenger pathways involved in the modulation of TTX-R I Na are still being defined. We have examined the modulation of TTX-R I Na in isolated sensory neurons with whole-cell voltage-clamp recording. Activation of either PKC or PKA increased TTX-R I Na . PKA activation also produced a leftward shift in the conductancevoltage relationship of TTX-R I Na and an increase in the rates of current activation, deactivation, and inactivation. Inhibitors of PKC decreased TTX-R I Na , whereas inhibitors of PKA had no effect on the current. Investigating the interaction between PKC and PKA revealed that although inhibitors of PKA had little effect on PKC-induced modulation of TTX-R I Na , inhibitors of PKC significantly attenuated PKA-induced modulation of the current. Finally, although PGE 2 -induced modulation of TTX-R I Na was more similar to PKA-induced modulation of the current than to PKC-induced modulation, PGE 2 -induced effects were inhibited by inhibitors of both PKC and PKA. Thus, although TTX-R I Na is a common target for cellular processes involving the activation of either PKA or PKC, PKC activity is necessary to enable subsequent PKA-mediated modulation of TTX-R I Na . Key words: dorsal root ganglion; inflammatory mediator; nociception; pain; primary afferent; second-messengerStudies of voltage-gated sodium currents (VGSC s) indicate that VGSC isoforms may be differentially modulated by protein kinase C (PKC) and protein kinase A (PK A). For example, a VGSC from brain tissue is decreased by the concurrent activation of PKC and PK A (Gershon et al., 1992;Li et al., 1992; Cantrell et al., 1996 Cantrell et al., , 1997, and a VGSC from cardiac muscle is decreased by PKC activation (Qu et al., 1994) and increased by PK A activation (Frohnwieser et al., 1995(Frohnwieser et al., , 1997. That these changes in VGSCs are physiologically relevant is suggested by the observations that receptor-mediated changes in cellular excitability reflect, at least in part, PKC -and /or PK A-mediated changes in VGSCs.We and others have recently demonstrated that a tetrodotoxinresistant voltage-gated Na ϩ current (TTX-R I Na ), expressed primarily in nociceptive afferents, is modulated by hyperalgesic inflammatory mediators in a manner that is likely to enhance nociceptor excitability (England et al., 1996;Gold et al., 1996b; Cardenas et al., 1997). Although there is evidence both for (England et al., 1996) and against (C ardenas et al., 1997) a role for protein kinase A in the modulation of TTX-R I Na , the contribution of PKC has yet to be investigated.Using agents that activate or inhibit PKC and PK A, we have tested the hypothesis that these kinases are involved in the modulation of T...
The voltage gated sodium channel comprises a pore-forming a subunit and regulatory b subunits. We report here the identification and characterization of a novel splicing variant of the human b 1 subunit, termed b 1B . The 807 bp open reading frame of the human b 1B subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human b 1B subunit shares an identical N-terminal half (residues 1-149) with the human b 1 subunit, but contains a novel C-terminal half (residues 150-268) of less than 17% sequence identity with the human b 1 subunit. The C-terminal region of the human b 1B is also significantly different from that of the rat b 1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human b 1B subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human b 1B subunit increases the ionic current when coexpressed with the tetrodotoxin sensitive channel, Na V 1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human b 1 and rat b 1A subunits.Keywords: sodium channel; b 1B subunit; splicing variant.By mediating the rapid entry of sodium ions into excitable cells in response to voltage changes across the plasma membrane, voltage gated sodium channels (VGSCs) play a fundamental role in the control of neuronal excitability in the central and peripheral nervous systems. The VGSC is a heteromeric protein complex that comprises at least a large (200-300 kDa) pore-forming a subunit and several smaller (30-40 kDa) regulatory b subunits [1][2][3][4]. It is well known that sodium channel a subunits determine the basic properties of the channel, while b subunits modulate the channel properties. Functional studies in a heterologous system have demonstrated that, depending on the type of coexpressed a subunit, b subunits are able to modulate almost all aspects of the channel properties, including voltage dependent gating, activation and inactivation, as well as greatly increasing the number of functional channels present on the plasma membrane [5,6]. Currently, at least nine different a subunits, three b subunits, and a splicing variant of the b 1 subunit, rat b 1A [7], have been cloned and characterized.The rat b 1A subunit is a splicing variant of the b 1 subunit via intron retention. The N-terminal half of the b 1A subunit is identical to that of the rat b 1 subunit, whereas its C-terminal half, encoded by a retained intron with an in-frame stop codon, is completely different from that of the rat b 1 subunit (to which it shows less than 17% identity). Coexpression of the rat b 1A subunit with the pore forming alpha subunit, Na V 1.2, in Chinese hamster lung 1610 cells, increased the sodium current density and produced subtle changes in voltage dependent activation and inactivation [7]. To further explore the function and physiological relevance of the sodium channel b 1 splicing...
The function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to Shaker potassium channels expressed in live Xenopus oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.
Volatile anesthetics inhibit mammalian voltage-gated Na ϩ channels, an action that contributes to their presynaptic inhibition of neurotransmitter release. We measured the effects of isoflurane, a prototypical halogenated ether volatile anesthetic, on the prokaryotic voltage-gated Na ϩ channel from Bacillus halodurans (NaChBac). Using whole-cell patch-clamp recording, human embryonic kidney 293 cells transfected with NaChBac displayed large inward currents (I Na ) that activated at potentials of Ϫ60 mV or higher with a peak voltage of activation of 0 mV (from a holding potential of Ϫ80 mV) or Ϫ10 mV (from a holding potential of Ϫ100 mV). Isoflurane inhibited I Na in a concentration-dependent manner over a clinically relevant concentration range; inhibition was significantly more potent from a holding potential of Ϫ80 mV (IC 50 ϭ 0.35 mM) than from Ϫ100 mV (IC 50 ϭ 0.48 mM). Isoflurane positively shifted the voltage dependence of peak activation, and it negatively shifted the voltage dependence of end steady-state activation. The voltage dependence of inactivation was negatively shifted with no change in slope factor. Enhanced inactivation of I Na was 8-fold more sensitive to isoflurane than reduction of channel opening. In addition to tonic block of closed and/or open channels, isoflurane enhanced use-dependent block by delaying recovery from inactivation. These results indicate that a prokaryotic voltage-gated Na ϩ channel, like mammalian voltage-gated Na ϩ channels, is inhibited by clinical concentrations of isoflurane involving multiple state-dependent mechanisms. NaChBac should provide a useful model for structure-function studies of volatile anesthetic actions on voltage-gated ion channels.
The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure–function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372–2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at −90 mV while holding at −150 mV. Charge–voltage (Q–V) curves showed sigmoidal dependence on voltage with gating charge saturating at −10 mV. Charge movement was shifted by −22 mV relative to the conductance–voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 μs observed for a change in preconditioning voltage from −160 to −80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q–V curves were shifted by approximately −60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.
The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.
Na + permeation through normal and batrachotoxin (BTX)-modified squid axon Na ÷ channels was characterized. Unmodified and toxin-modified Na ÷ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTXmodified channels were measured over a wide range of Na ÷ concentrations and voltages. Channel conductance as a function of Na + concentration curves showed that within the range 0.015-1 M Na ÷ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na + concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single-or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15°C, the estimated Q~0 for the conductance of the BTX-modified Na ÷ channel was 1.53. BTX appears not to change the Na ÷ channel ion selectivity (for the conditions used) or the surface charge located near the channel entrances.
Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.V oltage-gated sodium channels (Navs) play an essential role in the generation and propagation of action potentials in excitable cells (1). Eukaryotic Navs are composed of a poreforming α subunit and auxiliary β subunits. The α subunit is a large single-polypeptide chain organized in four different domains (DI-DIV), each of which has a voltage-sensing domain (VSD; S1-S4 segments) and a pore-forming domain (S5-S6 segments). Each domain has a different amino acid composition, pointing to some level of functional specialization. Site-directed fluorimetry shows that the VSDs in DI, DII, and DIII of the rat skeletal muscle voltage-gated sodium channel (Nav1.4) are activated by depolarization faster than in DIV (2). From this observation, it has been hypothesized that DI-III VSDs control the pore opening of the mammalian Nav, whereas the DIV VSD governs its fast inactivation (2-5).Although mammalian Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. The crystal structures of several prokaryotic Navs have been solved recently; however, in contrast to the mammalian Navs, they are homotetrameric, and thus structurally more closely related to the organization of voltage-gated potassium channels (Kvs) (6-9). The structure of a human L-type voltage-gated calcium channel type 1.1 (Cav1.1), which is a large single polypeptide composed of four different domains similar to mammalian Navs, has been resolved using cryoelectron microscopy (10, 11). However, functional studies have shown that gating mechanisms of mammalian Cav channels are indeed different from gating mechanisms in mammalian Navs (12). Furthermore, such structural studies only provide static snapshots of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are nee...
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