Voltage Dependence Of Slow Inactivation Research Articles

RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

Cav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel’s activation threshold toward more hyperpolarized potentials. Taken together, the association with “slow” β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

P1597Two novel mutations in SCN5A gene cause enhanced inactivation and lead to Brugada syndrome

Abstract Background Mutations in gene SCN5A, encoding cardiac potential-dependent sodium channel Nav1.5, are associated with various arrhythmogenic disorders among which the Brugada syndrome (BrS) and the Long QT syndrome (LQT) are the best characterized. BrS1 is associated with sodium channel dysfunction, which can be reflected by decreased current, impaired activation and enhanced inactivation. We found two novel mutations in our patients with BrS and explored their effect on fast and slow inactivation of cardiac sodium channel. Purpose The aim of this study was to investigate the effect of BrS (Y739D, L1582P) mutations on different inactivation processes in in vitro model. Methods Y739D and L1582P substitutions were introduced in SCN5A cDNA using site-directed mutagenesis. Sodium currents were recorded at room temperature in transfected HEK293-T cells using patch-clamp technique with holding potential −100 mV. In order to access the fast steady-state inactivation curve we used double-pulse protocol with 10 ms prepulses. To analyze voltage-dependence of slow inactivation we used two-pulse protocol with 10s prepulse, 20ms test pulse and 25ms interpulse at −100mV to allow recovery from fast inactivation. Electrophysiological measurements are presented as mean ±SEM. Results Y739D mutation affects highly conserved tyrosine 739 among voltage-gated sodium and calcium channels in the segment IIS2. Mutation L1582P located in the loop IVS4-S5, and leucine in this position is not conserved among voltage-gated channels superfamily. We have shown that Y739D leads to significant changes in both fast and slow inactivation, whereas L1582P enhanced slow inactivation only. Steady-state fast inactivation for Y739D was shifted on 8.9 mV towards more negative potentials compare with that for WT, while L1582P did not enhanced fast inactivation (V1/2 WT: −62.8±1.7 mV; Y739D: −71.7±2.3 mV; L1582P: −58.7±1.4 mV). Slow inactivation was increased for both substitutions (INa (+20mV)/INa (−100mV) WT: 0.45±0.03; Y739D: 0,34±0.09: L1582P: 0.38±0.04). Steady-state fast inactivation Conclusions Both mutations, observed in patients with Brugada syndrome, influence on the slow inactivation process. Enhanced fast inactivation was shown only for Y739D mutant. The more dramatic alterations in sodium channel biophysical characteristics are likely linked with mutated residue conservativity. Acknowledgement/Funding RSF #17-15-01292

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 μmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 μmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.

A Complex of RIM2alpha and RIM-Binding Protein 2 Stabilizes Slow Voltage-Dependent Inactivation of Cochlear Inner Hair Cell Cav1.3 L-Type Ca2+ Channels

Cav1.3 L-type Ca2+-channels (LTCCs) in cochlear inner hair cells (IHCs) exhibit a unique slow current inactivation to enable tonic sound-induced neurotransmitter release. The slow Ca2+-dependent inactivation can be explained by the calmodulin-competing binding of CaBP2, whereas the molecular mechanism underlying slow voltage-dependent inactivation (VDI) remains unexplained. Here we investigated if slow VDI is stabilized by the formation of a large signaling complex of Cav1.3 with the presynaptic scaffolding proteins RIM and RIM-binding-protein (RBP). The expression of the putative interaction partners in IHCs was investigated via nested-PCR. RBP2 interaction with the C-terminus of the full length (Cav1.3L) or short Cav1.3 isoform (Cav1.3S) was determined using GST-pulldowns and co-immunoprecipitations. The functional impact of coexpressed RIM and/or RBP on VDI of Cav1.3 Ba2+ currents (15 mM,β3 or β2a,α2δ1) was evaluated via whole-cell patch-clamp recordings in tsA201-cells. We detected RIM2α and RBP2+3 transcripts in mature IHCs and showed for the first time that β2e is the predominant IHC β2-splice variant. RBP2 could only interact with the full-length Cav1.3 C-terminus. Using β3, coexpression of RIM2α and RBP2 significantly slowed Cav1.3L VDI to a similar extent than observed in IHCs, while RIM2α or RBP2 alone had less (RIM2α) or no effect (RBP2). This effect was splice variant-specific because when co-expressed with Cav1.3S RBP2 even antagonized the slowing of VDI by RIM2α. The palmitoylated β2a-isoform slowed VDI by its own preventing an additional effect by RIM2α and/or RBP2 (β2e is currently investigated). We show that different mechanisms contribute to the slow VDI of IHCs. RIM2α/RBP2 stabilize slow VDI in Cav1.3L-β3 LTCC complexes while membrane-associated β2 variants (β2a and most probably also β2e) generate slow VDI without further modulation by RIM/RBP. Supported by Austrian Science Fund (FWFP27809,W11010,F44150).

Enhanced Slow Inactivation of the Human Skeletal Muscle Sodium Channel Causing Normokalemic Periodic Paralysis

Normokalemic periodic paralysis (normoPP) is a type of skeletal muscle function disorder which is characterized by paralysis attack with concomitant normal serum potassium level. We previously reported that R675Q mutation of human skeletal muscle voltage-gated sodium channel α subunit (SCN4A) may be the novel mutation which caused normoPP in Chinese families. However, it is still not clear how this mutation affects the SCN4A channel function. In this study, we used patch-clamp recording to study the function of wild type (WT) and R675Q mutant of SCN4A channels expressed in human embryonic kidney (HEK293) cells. We found that R675Q mutation did not affect the voltage dependence of sodium channel activation. The fast inactivation was also not significantly affected by R675Q mutation. However, R675Q mutation of SCN4A channels exhibited an 11.1 mV hyperpolarized shift in the voltage dependence of slow inactivation and significantly prolonged the recovery from prolonged inactivation state. Our results thus indicate that SCN4A was functionally affected by R675Q mutation, suggesting a possible reason for causing normoPP in Chinese patients.

Novel Mutations Mapping to the Fourth Sodium Channel Domain of Nav1.7 Result in Variable Clinical Manifestations of Primary Erythromelalgia

We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM.Electronic supplementary materialThe online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.

How Does KCNE1 Regulate the Kv7.1 Potassium Channel? Model-Structure, Mutations, and Dynamics of the Kv7.1-KCNE1 Complex

The voltage-gated potassium channel Kv7.1 and its auxiliary subunit KCNE1 are expressed in the heart and give rise to the major repolarization current. The interaction of Kv7.1 with the single transmembrane helix of KCNE1 considerably slows channel activation and deactivation, raises single-channel conductance, and prevents slow voltage-dependent inactivation. We built a Kv7.1-KCNE1 model-structure. The model-structure agrees with previous disulfide mapping studies and enables us to derive molecular interpretations of electrophysiological recordings that we obtained for two KCNE1 mutations. An elastic network analysis of Kv7.1 fluctuations in the presence and absence of KCNE1 suggests a mechanistic perspective on the known effects of KCNE1 on Kv7.1 function: slow deactivation is attributed to the low mobility of the voltage-sensor domains upon KCNE1 binding, abolishment of voltage-dependent inactivation could result from decreased fluctuations in the external vestibule, and amalgamation of the fluctuations in the pore region is associated with enhanced ion conductivity.

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

Role of the Local Anesthetic Receptor in the State-Dependent Inhibition of Voltage-Gated Sodium Channels by the Insecticide Metaflumizone

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Na(v)) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Na(v)1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Na(v)1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Na(v)1.4/F1579A and Na(v)1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Na(v)1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Na(v)1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class.

Signaling Complex of Calcium/Calmodulin-Dependent Protein Kinase II Associated with the C-Terminal Domain of Ca V2.1 Channels

Abstract:Ca+2/calmodulin-dependent protein kinase II (CaMKII) forms a major component of postsynaptic density, where its functions are well established but the presynaptic actions of CaMKII are poorly defined. We show that CaMKII constitutively binds and modulates presynaptic voltage-gated calcium (Cav2.1) channels that conduct P/Q type calcium currents. Using co-immunoprecipitation methods, we isolated a signaling complex of Cav2.1 with bound CaMKII. The pore-forming 1 subunit of Cav2.1 channels offers a unique platform for association of CaMKII at the C-terminal domain. The Cav2.1/CaMKII signaling complex operates as a network of multiprotein machines by recruiting other protein components and processing information by a series of protein interactions and protein phosphorylation reactions. Using GST pulldown assays we dissected the preferential binding of the inactive kinase to the Cav2.1 channel and mapped the binding site. Binding of CaMKII at this site per se is sufficient to slow voltage-dependent inactivation and shift the voltage dependence of inactivation to more positive membrane potentials, thereby greatly enhancing Cav2.1 channel activity. Using transfection assays, we show that the bound kinase is autophosphorylated at Thr286 and phosphorylates synapsin 1. We observed more than a six-fold increase in the synapsin 1 phosphorylation by CaMKII bound to the C-terminal domain Cav2.1 channel. Synapsin phosphorylation at Ser603 by CaMKII leads to formation of stable higher order oligomers. Our studies reveal a signaling complex of Cav2.1/CaMKII at the C-terminal domain of the Cav2.1 channel. Association of CaMKII with this signaling complex generates rapid response to calcium entry, recruitment of protein components, and phosphorylation of nearby proteins. Hence, kinase couples calcium influx to the downstream pathways for rapid, localized signal transduction and regulation.Supported by R01 NS22625

A new Na v1.7 sodium channel mutation I234T in a child with severe pain

Dominant gain-of-function mutations that hyperpolarize activation of the Na v1.7 sodium channel have been linked to inherited erythromelalgia (IEM), a disorder characterized by severe pain and redness in the feet and hands in response to mild warmth. Pharmacotherapy remains largely ineffective for IEM patients with cooling and avoidance of triggers being the most reliable methods to relieve pain. We now report a 5 year old patient with pain precipitated by warmth, together with redness in her hands and feet. Her pain episodes were first reported at 12 months, and by the age of 15–16 months were triggered by sitting as well as heat. Pain has been severe, inducing self-mutilation, with limited relief from drug treatment. Our analysis of the patient’s genomic DNA identified a novel Na v1.7 mutation which replaces isoleucine 234 by threonine (I234T) within domain I/S4–S5 linker. Whole-cell voltage-clamp analysis shows a I234T-induced shift of −18 mV in the voltage-dependence of activation, accelerated time-to-peak, slowed deactivation and enhanced responses to slow ramp depolarizations, together with a −21 mV shift in the voltage-dependence of slow-inactivation. Our data show that I234T induces the largest activation shift for Na v1.7 mutations reported thus far. Although enhanced slow-inactivation may attenuate the gain-of-function of the I234T mutation, the shift in activation appears to be dominant, and is consistent with the severe pain symptoms reported in this patient.

Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels

Cav1.4 channels are unique among the high voltage-activated Ca2+ channel family because they completely lack Ca2+-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca2+ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca2+-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca2+-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca2+ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca2+ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca2+-dependent inactivation, whereas it does not interfere with other calmodulin effects.

A novel dominant mutation of the Na v 1.4 α-subunit domain I leading to sodium channel myotonia

Mutations in SCN4A may lead to myotonia. Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Functional properties and differential neuromodulation of Na v1.6 channels

The voltage-gated sodium channel Na v1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Na V1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between Na V1.6 and Na V1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Na v1.6 channels, they conducted substantially larger persistent sodium currents than Na v1.2 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 v1.6 channels in neurons, was not observed for Na V1.6 expressed alone or with the auxiliary β 4 subunit. The unique properties of Na V1.6 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.

Slow voltage-dependent inactivation of L-type calcium channel CaV1.3 plays an important role in keeping its availability during pacemaker action potential.

Slow voltage-dependent inactivation of L-type calcium channel CaV1.3 plays an important role in keeping its availability during pacemaker action potential.

Mechanism of slow voltage-dependent inactivation of L-type Ca2+ channel, CaV1.3.

Mechanism of slow voltage-dependent inactivation of L-type Ca2+ channel, CaV1.3.

An Inner Pore Residue (Asn406) in the Nav1.5 Channel Controls Slow Inactivation and Enhances Mibefradil Block to T-Type Ca2+ Channel Levels

Mibefradil is a tetralol derivative once marketed to treat hyper-tension. Its primary target is the T-type Ca(2+) channel (IC(50), approximately 0.1-0.2 microM), but it also blocks Na(+),K(+),Cl(-), and other Ca(2+) channels at higher concentrations. We have recently reported state-dependent mibefradil block of Na(+) channels in which apparent affinity was enhanced when channels were recruited to slow-inactivated conformations. The structural determinants controlling mibefradil block have not been identified, although evidence suggests involvement of regions near or within the inner pore. We tested whether mibefradil interacts with the local anesthetic (LA) binding site, which includes residues in the S6 segments of domains (D) I, III, and IV. Mutagenesis of DIII S6 and DIVS6 did not reveal critical binding determinants. Substitution of Asn406 in DI S6 of cardiac Na(v)1.5, however, altered affinity in a manner dependent on the identity of the substituting residue. Replacing Asn406 with a phenylalanine or a cysteine increased affinity by 4- and 7-fold, respectively, thus conferring T-type Ca(2+) channel-like mibefradil sensitivity to the Na(+) channel. A series of other substitutions that varied in size, charge, and hydrophobicity had minimal effects on mibefradil block, but all mutations dramatically altered the magnitude and voltage-dependence of slow inactivation, consistent with data in other isoforms. Channels did not slow-inactivate, however, at the voltages used to assay mibefradil block, supporting the idea that Asn406 lies within or near the mibefradil binding site.

Cav1.4 Encodes a Calcium Channel with Low Open Probability and Unitary Conductance

When transiently expressed in tsA-201 cells, Cav1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100mM external barium as the charge carrier to determine the single-channel properties of Cav1.4 and to compare them to those of the Cav1.2. Cav1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Cav1.4 and Cav1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of −20 to +20mV. The single-channel conductance of Cav1.4 channels was ∼4pS compared with ∼20pS for Cav1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Cav1.4 channels pass cesium ions with a single-channel conductance of ∼21pS. Although Cav1.2 opening events were best described kinetically with two open time constants, Cav1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Cav1.4 channels by ∼45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Cav1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.

State-Dependent Block of Rat Na v1.4 Sodium Channels Expressed in Xenopus Oocytes by Pyrazoline-Type Insecticides

Insecticidal pyrazolines inhibit voltage-sensitive sodium channels of both insect and mammalian neurons in a voltage-dependent manner. Studies on the effects of pyrazoline insecticides on mammalian sodium channels have been limited to experimentation on the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channel populations of rat dorsal root ganglion (DRG) neurons. In this study, we examined the effects of the insecticidal pyrazolines indoxacarb, the N-decarbomethoxyllated metabolite of indoxacarb (DCJW), and RH 3421 on rat Na v1.4 sodium channels expressed in Xenopus laevis oocytes using the two-electrode voltage clamp technique. Both DCJW and RH 3421 were ineffective inhibitors of rat Na v1.4 sodium channels at a membrane potential of −120 mV, but depolarization to −60 mV or −30 mV during insecticide exposure resulted in substantial block. Inhibition by pyrazoline insecticides was nearly irreversible with washout, but repolarization of the membrane relieved block. DCJW and RH 3421 also caused hyperpolarizing shifts in the voltage dependence of slow inactivation without affecting the voltage dependence of activation or fast inactivation. These results suggest that DCJW and RH 3421 interact specifically with the slow inactivated state of the sodium channel. Indoxacarb did not cause block at any potential, yet it interfered with the ability of DCJW, but not RH 3421, to inhibit sodium current. Phenytoin, an anticonvulsant, reduced the efficacy of both DCJW and RH 3421. These data imply that the binding site for pyrazoline insecticides overlaps with that for therapeutic sodium channel blockers.

Differential Role of the α1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, β Subunits, and Ca2+ Ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.