Yusheng Qu

Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit.

α-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na+ channel α subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel α subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-α-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable α-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of α-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the α subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for α-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for α-scorpion and sea anemone toxins, respectively. Dissociation of α-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. α-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in α-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in α-scorpion toxin binding affinity between cardiac and neuronal Na+ channels. The results also support the hypothesis that this region of the Na+ channel is important for coupling channel activation to fast inactivation.

Voltage sensor-trapping

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

A sodium channel signaling complex: modulation by associated receptorprotein tyrosine phosphatase |[beta]|

Voltage-gated sodium channels in brain neurons were found to associate with receptor protein tyrosine phosphatase β (RPTPβ) and its catalytically inactive, secreted isoform phosphacan, and this interaction was regulated during development. Both the extracellular domain and the intracellular catalytic domain of RPTPβ interacted with sodium channels. Sodium channels were tyrosine phosphorylated and were modulated by the associated catalytic domains of RPTPβ. Dephosphorylation slowed sodium channel inactivation, positively shifted its voltage dependence, and increased whole-cell sodium current. Our results define a sodium channel signaling complex containing RPTPβ, which acts to regulate sodium channel modulation by tyrosine phosphorylation.

Modulation of Cardiac Na+ Channel Expression in Xenopus Oocytes by β1 Subunits

Voltage-gated Na+ channels consist of a large α subunit of 260 kDa associated with β1 and/or β2 subunits of 36 and 33 kDa, respectively. α subunits of rat cardiac Na+ channels (rH1) are functional when expressed alone in Xenopus oocytes or mammalian cells. β1 subunits are present in the heart, and localization of β1 subunit mRNA by in situ hybridization shows expression in the perinuclear cytoplasm of cardiac myocytes. Coexpression of β1 subunits with rH1 α subunits in Xenopus oocytes increases Na+ currents up to 6-fold in a concentration-dependent manner. However, no effects of β1 subunit coexpression on the kinetics or voltage dependence of the rH1 Na+ current were detected. Increased expression of Na+ currents is not observed when an equivalent mRNA encoding a nonfunctional mutant β1 subunit is coexpressed. Our results show that β1 subunits are expressed in cardiac muscle cells and that they interact with α subunits to increase the expression of cardiac Na+ channels in Xenopus oocytes, suggesting that β1 subunits are important determinants of the level of excitability of cardiac myocytes in vivo.

Differential modulation of sodium channel gating and persistent sodium currents by the β1, β2, and β3 subunits

Abstract Brain sodium channels are complexes of a pore-forming α subunit with auxiliary β subunits, which are transmembrane proteins that modulate α subunit function. The newly cloned β3 subunit is shown to be expressed broadly in neurons in the central and peripheral nervous systems, but not in glia and most nonneuronal cells. β1, β2, and β3 subunits are coexpressed in many neuronal cell types, but are differentially expressed in ventromedial nucleus of the thalamus, brain stem nuclei, cerebellar Purkinje cells, and dorsal root ganglion cells. Coexpression of β1, β2, and β3 subunits with Na v 1.2a α subunits in the tsA-201 subclone of HEK293 cells shifts sodium channel activation and inactivation to more positive membrane potentials. However, β3 is unique in causing increased persistent sodium currents. Because persistent sodium currents are thought to amplify summation of synaptic inputs, expression of this subunit would increase the excitability of specific groups of neurons to all of their inputs.

Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. β-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that β-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu779 in extracellular loop IIS1-S2 and both Glu837 and Leu840 in extracellular loop IIS3-S4 reduce the binding affinity of β-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect β-scorpion toxin binding but alter voltage dependence of activation and enhance β-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

Molecular mechanism of convergent regulation of brain Na(+) channels by protein kinase C and protein kinase A anchored to AKAP-15.

Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in hippocampal neurons voltage-dependent in a manner via phosphorylation of the alpha subunit. This modulation is dependent upon activation of cAMP-dependent protein kinase (PKA) and requires phosphorylation of serine 573 (S573) in the intracellular loop connecting homologous domains I and II (L(I-II)) by PKA anchored to A kinase anchoring protein-15 (AKAP-15). Activation of protein kinase C (PKC) also reduces peak Na(+) currents and enhances the strength of the PKA modulatory pathway. Here we probe the molecular mechanism responsible for the convergent effects of PKA and PKC on brain Na(v)1.2a channels. Analysis of the interaction of AKAP-15 with the intracellular loops of the Na(v)1.2a channel shows that it binds to L(I-II), thereby targeting PKA directly to its sites of phosphorylation on the Na(+) channel by specific protein-protein interactions. Mutagenesis and expression experiments indicate that reduction of peak Na(+) current by PKC requires S554 and S573 in L(I-II) in addition to S1506 in the inactivation gate. In addition, PKC-dependent phosphorylation of S576 in L(I-II) is necessary for enhancement of PKA modulation of brain Na(+) channels. When S576 is phosphorylated by PKC, the increase in modulation by PKA activation requires phosphorylation of S687 in L(I-II). Thus, the maximal modulation of these Na(+) channels by concurrent activation of PKA and PKC requires phosphorylation at four distinct sites in L(I-II): S554, S573, S576, and S687. This convergent regulation provides a novel mechanism by which information from multiple signaling pathways may be integrated at the cellular level in the hippocampus and throughout the central nervous system.

Functional roles of the extracellular segments of the sodium channel α subunit in voltage-dependent gating and modulation by β1 subunits

Voltage-gated sodium channels consist of a pore-forming α subunit associated with β1 subunits and, for brain sodium channels, β2 subunits. Although much is known about the structure and function of the α subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) α subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) α subunit. In comparison with rH1, wild-type rIIA α subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of β1 subunits inXenopus oocytes. When α subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA α subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA α subunits, all of the chimeric rIIA α subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with β1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of β1 subunits in shifting rIIA α subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by β1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.