Mutations of amino acid residues in the inner twothirds of the S6 segment in domain III of the rat brain type IIA Na ؉ channel (G1460A to I1473A) caused periodic positive and negative shifts in the voltage dependence of activation, consistent with an ␣-helix having one face on which mutations to alanine oppose activation. Mutations in the outer one-third of the IIIS6 segment all favored activation. Mutations in the inner half of IIIS6 had strong effects on the voltage dependence of inactivation from closed states without effect on open-state inactivation. Only three mutations had strong effects on block by local anesthetics and anticonvulsants. Mutations L1465A and I1469A decreased affinity of inactivated Na ؉ channels up to 8-fold for the anticonvulsant lamotrigine and its congeners 227c89, 4030w92, and 619c89 as well as for the local anesthetic etidocaine. N1466A decreased affinity of inactivated Na ؉ channels for the anticonvulsant 4030w92 and etidocaine by 3-and 8-fold, respectively, but had no effect on affinity of the other tested compounds. Leu-1465, Asn-1466, and Ile-1469 are located on one side of the IIIS6 helix, and mutation of each caused a positive shift in the voltage dependence of activation. Evidently, these amino acid residues face the lumen of the pore, contribute to formation of the high-affinity receptor site for pore-blocking drugs, and are involved in voltage-dependent activation and coupling to closed-state inactivation.

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 voltagedependent 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.electrical excitability ͉ gating E lectrical signaling in biology depends upon a unique electromechanical transduction process that couples small changes in the electrical potential across the cell membrane to conformational changes that open and close the pores of voltage-gated ion channels. The high sensitivity of voltage-gated ion channels to small changes in membrane potential depends on gating charges within their transmembrane structure, which are driven outward across the membrane by changes in the electric field and trigger a series of conformational changes that result in opening the pore (1-4). Approximately 12-16 positive charges move across the membrane during activation of the voltage sensors of voltage-gated sodium or potassium channels (5-10). Two major thermodynamic obstacles that the voltage-sensing process must overcome are stabilization of amino acid residues that serve as gating charges in the transmembrane environment of the protein and catalysis of their outward transmembrane movement.Voltage-gated ion channels are composed of four subunits or domains that each contain six transmembrane segments (11). The S1-S4 segments serve as the voltage sensing module, and the S5 and S6 segment and their connecting P loop serve as the pore-forming module (2-4). The primary gating charges reside in the S4 segment in four to seven repeated motifs of a positively charged amino acid residue (usually arginine) followed by two hydrophobic residues (2-4). The crux of the problem of understanding the electromechanical coupling in voltage-gated ion channels is determining the structural and mechanistic basis for movement of these gating charges across the membrane and for coupling of this movement to pore opening. The sliding helix or helical screw models posit that sequential formation of ion pairs with negatively charged amino acid residues in t...

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.

We have investigated the mechanism underlying the modulation of the cardiac L-type Ca 2؉ current by protein kinase C (PKC). Using the patch-clamp technique, we found that PKC activation by 4-␣-phorbol 12-myristate 13-acetate (PMA) or rac-1-oleyl-2-acetylglycerol (OAG) caused a substantial reduction in Ba 2؉ current through Ca v1.2 channels composed of ␣11.2, ␤1b, and ␣2␦1 subunits expressed in tsA-201 cells. In contrast, Ba 2؉ current through a cloned brain isoform of the Ca v1.2 channel (rbC-II) was unaffected by PKC activation. Two potential sites of PKC phosphorylation are present at positions 27 and 31 in the cardiac form of Ca v1.2, but not in the brain form. Deletion of N-terminal residues 2-46 prevented PKC inhibition. Conversion of the threonines at positions 27 and 31 to alanine also abolished the PKC sensitivity of Ca v1.2. Mutant Cav1.2 channels in which the threonines were converted singly to alanines were also insensitive to PKC modulation, suggesting that phosphorylation of both residues is required for PKC-dependent modulation. Consistent with this, mutating each of the threonines individually to aspartate in separate mutants restored the PKC sensitivity of Ca v1.2, indicating that a change in net charge by phosphorylation of both sites is responsible for inhibition. Our results define the molecular basis for inhibition of cardiac Ca v1.2 channels by the PKC pathway.

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