tsA201 Cells Research Articles

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

Connexin 43 mimetic peptide Gap26 confers protection to intact heart against myocardial ischemia injury

Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia-reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia-reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.

Molecular correlates of age-dependent seizures in an inherited neonatal-infantile epilepsy

Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused by mutations in the gene SCN2A encoding the voltage-gated Na(+) channel Na(V)1.2. We identified two novel SCN2A mutations causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal and an adult splice variant of the human Na(+) channel Na(V)1.2 expressed heterologously in tsA201 cells together with beta1 and beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na(+) current, accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental expression of Na(V)1.2 and of another voltage-gated Na(+) channel, Na(V)1.6, using immunohistochemistry and real-time reverse transcription-polymerase chain reaction in mouse brain slices. We found that Na(V)1.2 channels are expressed early in development at axon initial segments of principal neurons in the hippocampus and cortex, but their expression is diminished and they are gradually replaced as the dominant channel type by Na(V)1.6 during maturation. This finding provides a plausible explanation for the transient expression of seizures that occur due to a gain-of-function of mutant Na(V)1.2 channels.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Cav1.3 voltage-gated L-type Ca2+ channels. Their voltage- and Ca2+-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2α (RIM2α) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Cav1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the β-subunit of the channel complex and RIM-induced slowing of both Ca2+- and voltage-dependent inactivation of Cav1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Cav1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Cav1.3 gating kinetics in immature IHCs.

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABA(A)Rs, respectively) in the fluorescence-based FLIPR(TM) Membrane Potential assay. The compound inhibited the signaling of all GABA(A)R subtypes included in the study with high nanomolar/low micromolar IC(50) values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC(50) values at the homomeric alpha1 and alpha2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 microm. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA(A)R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha1 GlyR homology model revealed two distinct putative binding sites formed by the 6' M2 residues together with the 2' residues and the 10' and 13' residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor.

KV4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization

We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, KV4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of KV4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type KV4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed KV4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged KV4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged KV4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged KV4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1-S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1-S2 linker of KV4.2 suggests that other related channels may similarly be amenable to this tagging strategy.

Increased Coupled Gating of L-Type Ca 2+ Channels During Hypertension and Timothy Syndrome

L-Type (Cav1.2) Ca(2+) channels are critical regulators of muscle and neural function. Although Cav1.2 channel activity varies regionally, little is known about the mechanisms underlying this heterogeneity. To test the hypothesis that Cav1.2 channels can gate coordinately. We used optical and electrophysiological approaches to record Cav1.2 channel activity in cardiac, smooth muscle, and tsA-201 cells expressing Cav1.2 channels. Consistent with our hypothesis, we found that small clusters of Cav1.2 channels can open and close in tandem. Fluorescence resonance energy transfer and electrophysiological studies suggest that this coupling of Cav1.2 channels involves transient interactions between neighboring channels via their C termini. The frequency of coupled gating events increases in hypertensive smooth muscle and in cells expressing a mutant Cav1.2 channel that causes arrhythmias and autism in humans with Timothy syndrome (LQT8). Coupled gating of Cav1.2 channels may represent a novel mechanism for the regulation of Ca(2+) influx and excitability in neurons, cardiac, and arterial smooth muscle under physiological and pathological conditions.

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC.

Both Voltage- and Ca2+-Dependent Inactivation of Cav1.2 Ca2+ Channels are Suppresed in Myotubes, Likely Because of Triad Junction Proteins other than RyR1

To investigate the relationship between calcium channel and EC coupling functions of CaV1.1 in skeletal muscle, we have compared its behavior with that of the cardiac L-type channel, CaV1.2. Using expression in tsA-201 cells, we showed that substituting CaV1.1 IQ motif residues into CaV1.2 IQ caused a loss of calmodulin binding and calcium-dependent inactivation (JBC 283:29301-11, 2008), raising the possibility that inactivation is maladaptive for CaV1.1 function in muscle. We thus compared inactivation of CaV1.2 in tsA-201 cells and dysgenic myotubes (null for CaV1.1). For CaV1.2 in tsA-201 cells (co-expressed with α2δ1 and β2a), I50 ms/Ipeak (R50) at +20 mV was .69 in 10 Ca and .87 in 10 Ba, whereas R50 for CaV1.2 in dysgenic myotubes was .95 at +20 mV in 10 Ca, indicating that both voltage- and calcium-dependent inactivation were suppressed. In tsA-201 cells, co-expression of β1a (the predominant skeletal muscle isoform) did not significantly alter R50 values from those with β2a. Furthermore, R50 values were similar for CaV1.2 expressed in dysgenic myotubes and myotubes null for both CaV1.1 and RyR1, suggesting that some component of muscle triad junctions other than RyR1 is responsible for the suppression of inactivation. We thus expressed CaV2.1 in dysgenic myotubes because this neuronal channel, unlike CaV1.2, is not targeted to triad junctions (PNAS 95:1903-1908, 1998). Consistent with the hypothesis CaV2.1 channels showed significant voltage- and calcium-dependent inactivation in dysgenic myotubes which were similar to those of the channel expressed in tsA-201 cells. We are currently seeking to identify the components involved in suppressing inactivation of CaV1.2 in myotubes. Supported by AHA0190016G to JDO and NIH/NIAMS (AR055104) and MDA4319 to KGB.

Localized Calcineurin in Calcium- Dependent Inactivation of L-type Calcium Channels

The open probability of CaV1.2 L-type Ca2+ channels is enhanced by cAMP-dependent protein kinase (PKA), which is scaffolded to CaV1.2 channels by A-kinase anchoring proteins (AKAPs). CaV1.2 channels also undergo negative autoregulation via Ca2+-dependent inactivation (CDI). CDI relies upon binding of Ca2+/calmodulin (CaM) to an IQ motif in the carboxy tail of CaV1.2 L-type channels, a mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. In neurons, AKAP79/150 anchors both PKA and the Ca2+-activated phosphatase calcineurin (CaN) to CaV1.2 channels. Using transfected tsA201 cells or neurons, and tools such as the isolated calcineurin autoinhibitory peptide, over-expression of the catalytically-inactive CaNH151A mutant, and RNAi suppression of AKAP79, we have found that channel-linked CaM serves as a Ca2+ sensor for CaN, and that Ca2+/CaM-activated CaN participates in CDI by reversing channel enhancement by kinases such as PKA.We have also observed that I→E substitution in the IQ motif produces a mutant CaV1.2I/EQ channel that - when co-expressed with AKAP79 in tsA201 cells - unexpectedly exhibits ultra-fast inactivation. Ultra-fast inactivation is eliminated in Ca2+-free Na+ external solution, as well as by over-expression of the CaNH151A mutant or stimulation of PKA with forskolin. One interpretation is that the intact IQ motif's affinity for Ca2+/CaM limits the speed of CDI, and that reducing IQ affinity for Ca2+/CaM via I→E substitution allows CDI to proceed at a greatly speeded rate. FRET results with the CaV1.2I/EQ mutant or with AKAP79 lacking the CaN anchoring motif suggest that, during periods of elevated channel activity, the IQ-CaM and AKAP79-CaN interactions are both necessary for CaN-mediated reversal of current enhancement by PKA. In sum, our work supports a synthetic view fusing previous ideas regarding CaM and phosphorylation signaling in CDI.

Molecular Basis of a C Terminal Modulatory Mechanism in Cav1.3 Voltage-Gated Ca2+ Channels

We have previously discovered an intramolecular interaction between proximal- (PCRD) and distal C-terminal (DCRD) modulatory domains in human Cav1.3 L-type Ca2+ channels (LTCCs) which affects channel activation and inactivation gating properties (Singh et al 2008). This is present in the long (hCav1.342) but not a short (hCav1.342A) splice variant. Interestingly, this regulation has not been reported for rat Cav1.3 channel analogues (Xu and Lipscombe 2001). We systematically compared the functional properties of long and short Cav1.3 splice variants of mouse and rat with human channels after expression in tsA-201 cells using the whole-cell patch-clamp technique.

Disulfide Locking Reveals a Closed State Interaction Within the Voltage Sensor of NaChBac

S4 transmembrane segments of voltage-gated ion channels move outward upon depolarization initiating a conformational change that opens the pore. Formation of ion pairs between gating-charge-carrying arginine residues (R1-4) in S4 and negatively charged amino acid residues in neighboring transmembrane segments are thought to catalyze movement of S4. We have demonstrated three open-state interactions of gating charges R3 and R4 with negative charges D60 and E70 in NaChBac using the disulfide-locking method (DeCaen et al. PNAS 2008, 2009). Here, we studied cysteine pairs hypothesized to stabilize the resting state of the voltage sensor. Single mutations D60C or R113C (R1C) yield viable channels and INa, but the double mutant (D60C:R1C) is not functional when transfected into tsA201 cells. Confocal microscopy of cells transfected with D60C:R1C-eGFP revealed lack of membrane trafficking and sequestration to intracellular inclusions, whereas trafficking of singly mutated channels was comparable to WT. We also studied D60C:L112C which does not impair membrane trafficking but disulfide-locks in the closed state as assessed by whole-cell voltage clamp. This disulfide interaction is abolished by extracellular perfusion with reducing agents BME and TCEP. Evidently, the position of the S4 segment at negative potentials allows disulfide-locking of D60C and L112C, and S4 immobilization at this position maintains the voltage sensor in a resting conformation and keeps the central pore closed. These data suggest that the first gating charge forms an ion pair with D60 in a resting state and their side chains approach within ∼2 A, as required for formation of disulfide bonds. These new molecular interactions allow further refinement of the ROSETTA sliding helix model of NaChBac gating (see poster by Yarov-Yarovoy).Supported by NIH Grants T32 GM07270 and R01 NS15751.

Gamma1 Subunit Renders Cav1.2 Channels Dependent on Cell Cycle

Auxiliary γ subunits are known to enhance inactivation of L-type calcium channels. When co-expressed in tsA-201 cells with α1C and β2a subunits, the γ1 subunit shifts the voltage-dependence of inactivation by about −20 mV. The voltage of half-maximal inactivation, V1/2, was determined with 5 s long conditioning pre-pulses. On average, addition of γ1 to α1C/β2a channels changed V1/2 from −24±5 mV (n=30) to −44±12 mV (n=92). We noticed that V1/2, but not the steepness of the voltage-dependence of inactivation, varies greatly in cells with γ1 and set up to find the cause of the cell-to-cell variability. Serum starvation and “ER shock” by the N-glycosylation blocker tunicamicin further shifted inactivation to negative voltages. The average V1/2 was −59±12 mV (n=12) in serum-free and −69±13 mV(n=32) in tunicamicin treated cells. These treatments altered inactivation only when γ1 was present and the effects were similar when β3 substituted for β2a. Mutations of γ1 that remove consensus N-glycosylation sites had only partial effect (V1/2=-60±18 mV, n=29) and did not reduce the cell-to-cell variability, indicating that N-glycosylation of γ1 was not its primary cause.Serum starvation and tunicamicin are known to produce cell-cycle arrest in the G0/G1 phase and, therefore, could act on γ1 indirectly by interfering with a cell-cycle dependent pathway. We characterized inactivation in cells expressing fluorescent probes visualizing cell-cycle activity. In support of our hypothesis, V1/2 was −55±16 mV, n=32, in G1 and −36±7 mV, n=20, in S/G2/M cells.Therefore, we propose that a novel cell-cycle dependent regulatory pathway controls voltage-dependent inactivation and functional availability of L-type calcium channels in the presence of γ1 subunit.The project was supported by MH079406 from NIMH.

Sodium Channel Variants Associated with Atrial Fibrillation Exhibit Abnormal Fast and Slow Inactivation

Mutations and rare genetic variants in SCN5A, the gene encoding the cardiac voltage-gated sodium channel NaV1.5, have been associated with inherited predisposition to ventricular arrhythmia. More recently, SCN5A variants have been identified in families segregating atrial fibrillation. We evaluated the biophysical properties of seven novel SCN5A variants associated with atrial fibrillation identified by our previous genetic study to elucidate potential molecular mechanisms underlying this common arrhythmia. Functional properties of E428K, H445D, N470K, E655K, T1131I, R1826C and V1951M were assessed by whole-cell patch clamp recording of recombinant mutant channels heterologously expressed with the human β1 subunit in tsA201 cells. One variant (R1826C) did not exhibit substantial differences in biophysical properties of activation or fast inactivation, and another variant (E655K) only exhibited minor differences in recovery from inactivation as compared with wildtype (WT) channels. However, two mutants (H445D, T1131I) exhibited significant shifts in the voltage-dependence of activation toward more negative potentials (p < 0.005), and four other mutant channels (E428K, H445D, N470K, V1951M) exhibited significant shifts in the voltage-dependence of steady-state inactivation toward more positive potentials as compared with WT channels. Further, H445D and V1951M exhibited more rapid onset, impaired recovery and enhancement of slow inactivation evoked by 1000 ms depolarizing prepulses as compared with WT channels. For the five variants with either hyperpolarized activation voltage-dependence or depolarized steady-state inactivation, we predict increased window current as defined by the overlap of these two curves. Increased window current and enhanced slow inactivation of some variants is further predicted to alter excitability and/or conduction in myocardial tissue and is a plausible mechanism by which SCN5A variants may increase vulnerability to atrial fibrillation.

In Vitro Cardiac Safety Profiling of a Novel Benzyl-Ethylamine Compound

When developing novel compounds for any clinical indication, the possibility of untoward cardiovascular effects must be addressed. To evaluate the cardiac electrophysiologic effects of A-674563 ((S)-1-benzyl-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine), a novel benzyl-ethylamine, we profiled the compound in a series of cellular and tissue assays. In canine Purkinje fibers (30 min exposure; 2 sec BCL), A-674563 elicited concentration dependent depolarization with shouldering of the terminal repolarization phase (20 μM) and increased abnormal automaticity (60 μM). In papillary muscles, concentration dependent depolarization was also seen, but the effect was much less potent; 60 μM induced shouldering of the terminal phase of repolarization. Contractility was assessed using percent changes in fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. A-674563 reduced FS in a concentration dependent manner; 10% at 2 μM and 47% at 20 μM. Effects on ionic currents were further evaluated using heterologously expressed cardiac ion channel cell lines. A-674563 inhibited Cav1.2, expressed in HEK cells, with an IC50 of 20 μM, suggesting that the decreased contractility seen in native cells is due to L-type calcium channel block. The compound reduced Nav1.5 (HEK cells) by 50% at 20 μM and inhibited Kir2.1 (tSA201 cells) with an IC50 of 35 μM. Block of these channels would be expected to reduce the upstroke velocity, depolarize the cellular membrane and lead to abnormal automaticity as was seen in the tissue assays. Although A-674563 caused minimal prolongation of action potential duration in tissues up to 60 μM, it inhibited hERG (HEK cells) with an IC50 of 0.7 μM. This potent block was most likely offset by the concomitant block of multiple cardiac ion channels. In conclusion, A-674563 affects multiple cardiac ion channels to elicit depolarization and increased automaticity in native cardiac tissues.

Regulation of the Chloride/Proton Exchanger CLC-5 By Internal pH

ClC-5 transports anions and protons across intracellular membranes and is necessary for endosomal/lysosomal acification. Similar to other ClC transporters, ClC-5 can switch between two different transport modes and operate either as anion-proton exchanger or as anion channel, depending on the type of external anion. So far, only some regulatory mechanisms that affect the functional mode of these transporters have been described. We here use combined whole-cell patch clamp and fluorescent intracellular pH recordings in transfected tsA201 cells to characterize ClC-5 mediated transport under various ionic conditions.

Divergent Pharmacological Properties of SCN1A Splice Variants

Voltage-gated sodium channels undergo alternative mRNA splicing. In the human neuronal Nav1.1 channel encoded by SCN1A, a common genetic variant affecting an intron splice donor site alters the proportion of transcripts that incorporate the canonical exon 5 (exon 5A) or an alternative (exon 5N) encoding portions of the S3 and S4 segments of domain 1. Epileptic subjects with this genetic variant require lower doses of anticonvulsant drugs such as phenytoin compared with individuals lacking this variant. Because this genetic variant is associated with a larger proportion of exon 5N containing transcripts in brain, we hypothesized that differences in function and pharmacology of Nav1.1 channels containing either exon 5N or 5A account for the observed divergence in anticonvulsant dose requirements. To examine differences in drug efficacy of SCN1A splice variants, we performed whole-cell recording on tsA201 cells transiently co-transfected with either Nav1.1-5A or Nav1.1-5N and two accessory subunits (β1,β2). We examined voltage-dependence of activation, steady-state inactivation, and recovery from fast inactivation and observed no significant differences between splice variants. We also measured both steady-state block and use-dependent block (10Hz) by phenytoin, carbamazepine, and lamotrigine. Nav1.1-5N channels exhibited greater steady-state block by phenytoin(100μM) (16±5% vs. 2±6%) and lamotrigine(200μM) (25±4% vs. 14±2%) compared to Nav1.1-5A. Additionally, Nav1.1-5N exhibited greater use-dependent block by phenytoin (39±5% vs. 24±4%) and lamotrigine (29±6% vs. 18±2%). We tested cells stably transfected with either Nav1.1-5A or Nav1.1-5N and both β subunits using an automated planar patch clamp system (Patchliner, Nanion Inc.) to perform concentration-response curves to determine steady-state and inactivated state affinities for lamotrigine. Similar to conventional patch clamp experiments, lamotrigine exhibited greater steady-state and inactivated state affinity for Nav1.1-5N than Nav1.1-5A. These results suggest SCN1A transcripts containing the alternative exon 5N encode channels that are more sensitive to multiple anticonvulsant drugs.

Reconstitution of PKA-Dependent Modulation of Cardiac Cav1.2 Channels

L-type calcium currents through Cav1.2 channels initiate contraction in cardiac muscle. Their regulation by neurotransmitters and hormones through second messenger signaling cascades and protein kinase A (PKA) phosphorylation is a key controller of calcium signaling and contractile force. The α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminal domain is proteolytically cleaved but reassociates non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of autoinhibition by cellular regulatory signals acting on the C-terminus provides an attractive mechanism for producing the large increases in calcium current that are observed physiologically. In fact, consistent reconstitution of PKA-dependent regulation of Cav1.2 channels in non-muscle has been difficult to achieve. To reconstitute such PKA regulation, we optimized the expression of truncated Cav1.2 channels, the distal C-terminal domain, the α2δ subunit, and the β2b subunit to give a functional autoregulatory complex as assessed by whole-cell voltage clamp recordings of tsA-201 cells. Expression of the truncated Cav1.2 channel with the free distal domain resulted in large decreases in inward barium current and in coupling between voltage-dependent gating and pore opening. We hypothesized that AKAP15 expression would promote PKA association with the distal C-terminal of the channel and increase the likelihood of PKA-dependent phosphorylation. After optimizing AKAP15 expression, currents recorded in 5 μM forskolin were approximately 5-fold larger than those recorded in the presence of kinase inhibitor RO 31-8220 (1 uM). These findings show that the full-range of PKA-dependent modulation of Cav1.2 channels can be reproduced when an autoinhibitory complex is formed in this manner and provide a substrate for further studies of this physiologically important regulatory process.Supported by NIH grant HL007312-31 and AHA fellowship 09POST2080270.

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, mu-, delta-, and kappa-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The mu-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of mu-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.

Membrane Topology of S4 of the Mouse Voltage-Gated Proton Channel

VSOP/Hv1 is a voltage-gated proton channel that contains the voltage sensor domain (VSD) but not pore domain. VSD of VSOP/Hv1 allows protons to permeate as well as sensing voltage. It has been reported that basic amino acids in the fourth transmembrane segment (S4) of voltage-gated ion channels play critical roles in voltage-sensing. Mouse VSOP (mVSOP) has three arginine residues (R1, R2, R3) in a pattern similar to those conserved in other voltage-gated channels. To address the role of S4 in mVSOP, we have reported that the truncated construct (A206sop) just downstream of R2 in the S4 is still ion-conductive (Biophysical Society 53th Annual Meeting, 2009). In this study, we further analysed properties of A206stop. The outward current of A206stop was almost completely blocked by zinc. Visualization of intracellular pH using BCECF (pH-sensitive ratiometric dye) showed that cytoplasm of tsA201 cell was alkalinized under the depolarization condition. Na and K ion do not permeate through A206stop. Gating properties of the proton currents through A206stop were sensitive to either intracellular pH or extracellular pH. However, voltage dependency of A206stop was weaker than that of full-length mVSOP, and the I-V relationship of A206stop was shifted rightward. These results indicate that A206stop retains the basic properties of the voltage-gated proton channel even if it lacks a half of S4. We also carried out two biochemical assays: site-directed cysteine-scanning using accessibility of maleimide-reagent as detected by western blotting (pegylation protection) and in vitro glycosylation assays. Both showed that S4 of A206stop inserts into the membrane and the position of A206 faces intracellular aqueous environments. These findings suggest that the region downstream of the R2 position of S4 of VSOP/Hv1 is not essential for proton selectivity.