NaV1.9 current in muscle afferent neurons is enhanced by substances released during muscle activity.

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

KV7 channels are potential regulators of the exercise pressor reflex.

The exercise pressor reflex (EPR) originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease or heart failure to increase the incidence of myocardial infarction. We seek to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Bradykinin (BK) is released within contracting muscle and can evoke the EPR. However, the mechanism is incompletely understood. KV7 channels strongly regulate neuronal excitability and are inhibited by BK. We have identified KV7 currents in muscle afferent neurons by their characteristic activation/deactivation kinetics, enhancement by the KV7 activator retigabine, and block by KV7 specific inhibitor XE991. The blocking of KV7 current by different XE991 concentrations suggests that the KV7 current is generated by both KV7.2/7.3 (high affinity) and KV7.5 (low affinity) channels. The KV7 current was inhibited by 300 nM BK in neurons with diameters consistent with both group III and group IV afferents. The inhibition of KV7 by BK could be a mechanism by which this metabolic mediator generates the EPR. Furthermore, our results suggest that KV7 channel activators such as retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease.NEW & NOTEWORTHY KV7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that KV7 current is generated by KV7.2/3 and KV7.5 channels. The BK inhibition of KV7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance KV7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.

Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.

Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.

NaV1.9 channels in muscle afferent neurons and axons.

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, NaV1.6, and NaV1.7) and -resistant (TTX-R, NaV1.8, and NaV1.9) voltage-gated sodium (NaV) channels, but NaV1.9 current has not yet been demonstrated. Using a F--containing internal solution, we found a NaV current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from NaV1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following NaV1.9 current rundown (internal F-), application of the NaV1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl-), which suggests that NaV1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were NaV1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also NaV1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of NaV1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel NaV1.9 plays a unique role in controlling afferent excitability. We show that NaV1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist.

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.

EXPRESS: Voltage-dependent sodium (NaV) channels in group IV sensory afferents.

Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (NaV) channels. We previously found tetrodotoxin-resistant NaV1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of NaV1.8 channels. To address these apparent differences in NaV channel expression between axon and soma, we used immunohistochemistry to identify the NaV channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express NaV1.8, NaV1.6, or NaV1.7. Interestingly, expression of NaV1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either NaV1.6 or NaV1.7. When combined with previous work, our results suggest that NaV1.8 channels are expressed in most group IV axons, but that, under normal conditions, NaV1.6 and/or NaV1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.

Functional expression of α7-nicotinic acetylcholine receptors by muscle afferent neurons.

The exercise pressor reflex (EPR) is generated by group III and IV muscle afferents during exercise to increase cardiovascular function. Muscle contraction is triggered by ACh, which is metabolized into choline that could serve as a signal of exercise-induced activity. We demonstrate that ACh can induce current in muscle afferents neurons isolated from male Sprague-Dawley rats. The nicotinic ACh receptors (nAChRs) appear to be expressed by some group III-IV neurons since capsaicin (TRPV1) and/or ATP (P2X) induced current in 56% of ACh-responsive neurons. α7- And α4ß2-nAChRs have been shown to be expressed in sensory neurons. An α7-nAChR antibody stained 83% of muscle afferent neurons. Functional expression was demonstrated by using the specific α7-nAChR blockers α-conotoxin ImI (IMI) and methyllycaconitine (MLA). MLA inhibited ACh responses in 100% of muscle afferent neurons, whereas IMI inhibited ACh responses in 54% of neurons. Dihydro-ß-erythroidine, an α4ß2-nAChR blocker, inhibited ACh responses in 50% of muscle afferent neurons, but recovery from block was not observed. Choline, an α7-nAChR agonist, elicited a response in 60% of ACh-responsive neurons. Finally, we demonstrated the expression of α7-nAChR by peripherin labeled (group IV) afferent fibers within gastrocnemius muscles. Some of these α7-nAChR-positive fibers were also positive for P2X3 receptors. Thus choline could serve as an activator of the EPR by opening α7-nAChR expressed by group IV (and possible group III) afferents. nAChRs could become pharmacological targets for suppressing the excessive EPR activation in patients with peripheral vascular disease.

Identification of CaV channel types expressed in muscle afferent neurons.

Cardiovascular adjustments to exercise are partially mediated by group III/IV (small to medium) muscle afferents comprising the exercise pressor reflex (EPR). However, this reflex can be inappropriately activated in disease states (e.g., peripheral vascular disease), leading to increased risk of myocardial infarction. Here we investigate the voltage-dependent calcium (CaV) channels expressed in small to medium muscle afferent neurons as a first step toward determining their potential role in controlling the EPR. Using specific blockers and 5 mM Ba(2+) as the charge carrier, we found the major calcium channel types to be CaV2.2 (N-type) > CaV2.1 (P/Q-type) > CaV1.2 (L-type). Surprisingly, the CaV2.3 channel (R-type) blocker SNX482 was without effect. However, R-type currents are more prominent when recorded in Ca(2+) (Liang and Elmslie 2001). We reexamined the channel types using 10 mM Ca(2+) as the charge carrier, but results were similar to those in Ba(2+). SNX482 was without effect even though ∼27% of the current was blocker insensitive. Using multiple methods, we demonstrate that CaV2.3 channels are functionally expressed in muscle afferent neurons. Finally, ATP is an important modulator of the EPR, and we examined the effect on CaV currents. ATP reduced CaV current primarily via G protein ßγ-mediated inhibition of CaV2.2 channels. We conclude that small to medium muscle afferent neurons primarily express CaV2.2 > CaV2.1 ≥ CaV2.3 > CaV1.2 channels. As with chronic pain, CaV2.2 channel blockers may be useful in controlling inappropriate activation of the EPR.

Domain III regulates N-type (CaV2.2) calcium channel closing kinetics.

Ca(V)2.2 (N-type) and Ca(V)1.2 (L-type) calcium channels gate differently in response to membrane depolarization, which is critical to the unique physiological functions mediated by these channels. We wondered if the source for these differences could be identified. As a first step, we examined the effect of domain exchange between N-type and L-type channels on activation-deactivation kinetics, which were significantly different between these channels. Kinetic analysis of chimeric channels revealed N-channel-like deactivation for all chimeric channels containing N-channel domain III, while activation appeared to be a more distributed function across domains. This led us to hypothesize that domain III was an important regulator of N-channel closing. This idea was further examined with R-roscovitine, which is a trisubstituted purine that slows N-channel deactivation by exclusively binding to activated N-channels. L-channels lack this response to roscovitine, which allowed us to use N-L chimeras to test the role of domain III in roscovitine modulation of N-channel deactivation. In support of our hypothesis, all chimeric channels containing the N-channel domain III responded to roscovitine with slowed deactivation, while those chimeric channels with L-channel domain III did not. Thus a combination of kinetic and pharmacological evidence supports the hypothesis that domain III is an important regulator of N-channel closing. Our results support specialization of gating functions among calcium channel domains.

Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons.

Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.

Roscovitine inhibits CaV3.1 (T-type) channels by preferentially affecting closed-state inactivation.

T-type calcium channels (Ca(V)3) play an important role in many physiological and pathological processes, including cancerogenesis. Ca(V)3 channel blockers have been proposed as potential cancer treatments. Roscovitine, a trisubstituted purine, is a cyclin-dependent kinase (CDK) inhibitor that is currently undergoing phase II clinical trials as an anticancer drug and has been shown to affect calcium and potassium channel activity. Here, we investigate the effect of roscovitine on Ca(V)3.1 channels. Ca(V)3.1 channels were transiently expressed in human embryonic kidney 293 cells, and currents were recorded by using the whole-cell patch-clamp technique. Roscovitine blocks Ca(V)3.1 channels with higher affinity for depolarized cells (EC50 of 10 µM), which is associated with a negative shift in the voltage dependence of closed-state inactivation. Enhanced inactivation is mediated by roscovitine-induced acceleration of closed-state inactivation and slowed recovery from inactivation. Small effects of roscovitine were also observed on T-channel deactivation and open-state inactivation, but neither could explain the inhibitory effect. Roscovitine inhibits Ca(V)3.1 channels within the therapeutic range (10-50 µM) in part by stabilizing the closed-inactivated state. The ability of roscovitine to block multiple mediators of proliferation, including CDKs and Ca(V)3.1 channels, may facilitate its anticancer properties.

Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation.

L-type (Ca(V)1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N- and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes.

Interference between two modulators of N-type (CaV2.2) calcium channel gating demonstrates that omega-conotoxin GVIA disrupts open state gating.

N-type calcium channels play an important role in synaptic transmission and a drug that blocks these channels has become an important tool in controlling chronic pain. The development of new N-channel-targeted drugs is dependent on a better understanding of the gating of these channels and how that gating can be modulated. We have previously concluded that omega-conotoxin GVIA (GVIA) is a gating modifier that acts by destabilizing the N-channel open state. However, this conclusion was largely based on our modeling results and requires experimental support. Roscovitine, a tri-substituted purine, has been shown to stabilize the N-channel open state to slow gating charge relaxation, which provides a direct test of our hypothesis for GVIA-induced gating modification. We found that roscovitine could modulate gating current in the presence of GVIA, which shows that roscovitine can still affect the gating of the GVIA-bound N-channel. However, the magnitude of the roscovitine-induced slowing of Off-gating current was significantly reduced. In addition to confirming our hypothesis, our evidence supports an additional effect of GVIA to alter gating transitions between N-channel closed states. By strongly limiting access to the N-channel open state, GVIA analogs that selectively induce this modulation could provide the basis for the next generation drugs that treat chronic pain.

Phospholemman modulates the gating of cardiac L-type calcium channels.

Ca(2+) entry through L-type calcium channels (Ca(V)1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca(V)1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca(V)1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca(V)1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca(2+)-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca(2+) influx via Ca(V)1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca(V)1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca(2+) overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca(2+) influx during the cardiac action potential waveform plateau phase.

Ceramide modulates HERG potassium channel gating by translocation into lipid rafts.

Human ether-à-go-go-related gene (HERG) potassium channels play an important role in cardiac action potential repolarization, and HERG dysfunction can cause cardiac arrhythmias. However, recent evidence suggests a role for HERG in the proliferation and progression of multiple types of cancers, making it an attractive target for cancer therapy. Ceramide is an important second messenger of the sphingolipid family, which due to its proapoptotic properties has shown promising results in animal models as an anticancer agent. Yet the acute effects of ceramide on HERG potassium channels are not known. In the present study we examined the effects of cell-permeable C(6)-ceramide on HERG potassium channels stably expressed in HEK-293 cells. C(6)-ceramide (10 microM) reversibly inhibited HERG channel current (I(HERG)) by 36 +/- 5%. Kinetically, ceramide induced a significant hyperpolarizing shift in the current-voltage relationship (DeltaV(1/2) = -8 +/- 0.5 mV) and increased the deactivation rate (43 +/- 3% for tau(fast) and 51 +/- 3% for tau(slow)). Mechanistically, ceramide recruited HERG channels within caveolin-enriched lipid rafts. Cholesterol depletion and repletion experiments and mathematical modeling studies confirmed that inhibition and gating effects are mediated by separate mechanisms. The ceramide-induced hyperpolarizing gating shift (raft mediated) could offset the impact of inhibition (raft independent) during cardiac action potential repolarization, so together they may nullify any negative impact on cardiac rhythm. Our results provide new insights into the effects of C(6)-ceramide on HERG channels and suggest that C(6)-ceramide can be a promising therapeutic for cancers that overexpress HERG.

Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.

Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

A single amino acid change in Ca(v)1.2 channels eliminates the permeation and gating differences between Ca(2+) and Ba(2+).

Glutamate scanning mutagenesis was used to assess the role of the calcicludine binding segment in regulating channel permeation and gating using both Ca(2+) and Ba(2+) as charge carriers. As expected, wild-type Ca(V)1.2 channels had a Ba(2+) conductance ~2x that in Ca(2+) (G(Ba)/G(Ca) = 2) and activation was ~10 mV more positive in Ca(2+) vs. Ba(2+). Of the 11 mutants tested, F1126E was the only one that showed unique permeation and gating properties compared to the wild type. F1126E equalized the Ca(V)1.2 channel conductance (G(Ba)/G(Ca) = 1) and activation voltage dependence between Ca(2+) and Ba(2+). Ba(2+) permeation was reduced because the interactions among multiple Ba(2+) ions and the pore were specifically altered for F1126E, which resulted in Ca(2+)-like ionic conductance and unitary current. However, the high-affinity block of monovalent cation flux was not altered for either Ca(2+) or Ba(2+). The half-activation voltage of F1126E in Ba(2+) was depolarized to match that in Ca(2+), which was unchanged from that in the wild type. As a result, the voltages for half-activation and half-inactivation of F1126E in Ba(2+) and Ca(2+) were similar to those of wild-type in Ca(2+). This effect was specific to F1126E since F1126A did not affect the half-activation voltage in either Ca(2+) or Ba(2+). These results indicate that residues in the outer vestibule of the Ca(V)1.2 channel pore are major determinants of channel gating, selectivity, and permeation.

Open-state occupancy prevents gating charge relaxation of N-type (CaV2.2) calcium channels.

N-type and L-type channels have significant gating differences, and we wondered whether some of these differences are linked to the relationship between charge movement and channel opening. The time constants for N-channel closing (tau(Deact)) and Off-gating charge movement (tauQ(Off)) were compared over a range of voltages. tauQ(Off) was significantly larger than tau(Deact) at voltages < -10 mV, and the voltage dependence of the tauQ(Off) was less steep than that for tau(Deact), which suggests that gating charge relaxation does not limit channel closing. Roscovitine, a drug that slows N-channel closing by holding the channel in a high open-probability state, was found to slow both tauQ(Off) and tau(Deact), and thus the time courses of channel closing and gating charge relaxation were similar. Our gating current results were reproduced with the addition of a voltage-independent, closed-closed transition to our previously published two-open-state N-channel model. This work suggests that, like L-type channels, there is a voltage-independent transition along the N-channel activation/deactivation pathway, but this transition occurs between closed states instead of the closed-open states of the L-channel. Also unlike L-type channels, the gating charge appears to be locked into the activated position by the N-channel open state.

The Timothy syndrome mutation of cardiac CaV1.2 (L-type) channels: multiple altered gating mechanisms and pharmacological restoration of inactivation.

Timothy syndrome (TS) is a multiorgan dysfunction caused by a Gly to Arg substitution at position 406 (G406R) of the human CaV1.2 (L-type) channel. The TS phenotype includes severe arrhythmias that are thought to be triggered by impaired open-state voltage-dependent inactivation (OSvdI). The effect of the TS mutation on other L-channel gating mechanisms has yet to be investigated. We compared kinetic properties of exogenously expressed (HEK293 cells) rabbit cardiac L-channels with (G436R; corresponding to position 406 in human clone) and without (wild-type) the TS mutation. Our results surprisingly show that the TS mutation did not affect close-state voltage-dependent inactivation, which suggests different gating mechanisms underlie these two types of voltage-dependent inactivation. The TS mutation also significantly slowed activation at voltages less than 10 mV, and significantly slowed deactivation across all test voltages. Deactivation was slowed in the double mutant G436R/S439A, which suggests that phosphorylation of S439 was not involved. The L-channel agonist Bay K8644 increased the magnitude of both step and tail currents, but surprisingly failed to slow deactivation of TS channels. Our mathematical model showed that slowed deactivation plus impaired OSvdI combine to synergistically increase cardiac action potential duration that is a likely cause of arrhythmias in TS patients. Roscovitine, a tri-substituted purine that enhances L-channel OSvdI, restored TS-impaired OSvdI. Thus, inactivation-enhancing drugs are likely to improve cardiac arrhythmias and other pathologies afflicting TS patients.