ABSTRACT
Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed "gating charges," which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltage-dependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Sodium Channels/chemistry , Sodium Channels/metabolism , Amino Acid Substitution , Bacterial Proteins/genetics , Disulfides/chemistry , Electrophysiological Phenomena , Ion Channel Gating , Kinetics , Models, Biological , Models, Molecular , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Sodium Channels/geneticsABSTRACT
The voltage-gated sodium channel Na(v)1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Na(V)1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between Na(V)1.6 and Na(V)1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Na(v)1.6 channels, they conducted substantially larger persistent sodium currents than Na(v)1.2 channels, and they were much less sensitive to inhibition by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Na(v)1.6 channels in neurons, was not observed for Na(V)1.6 expressed alone or with the auxiliary beta(4) subunit. The unique properties of Na(V)1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.
Subject(s)
Action Potentials/physiology , Nerve Tissue Proteins/physiology , Sodium Channels/physiology , Animals , Cell Line , Humans , Mice , NAV1.6 Voltage-Gated Sodium Channel , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Rats , Receptors, Neurotransmitter/physiology , Sodium Channels/chemistry , Sodium Channels/metabolismABSTRACT
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.