Regulation Of Channel Activity Research Articles

Inhibition of RyR2 from Failing and Non-Failing Human Hearts by Calmodulin

The RyR2 is a macromolecular complex comprising a Ca2+ channel and many other accessory proteins that regulate channel activity. The binding of intracellular calmodulin (CaM, ∼45 nM in the cytoplasm) partially inhibits calcium release in cardiomyocytes due to its ability to inhibit RyR2 channel opening. Single channel studies of RyR2 in lipid bilayers find that CaM inhibits RyR2 with an IC50 of 100 nM. During the process of RyR2 isolation fromthe heart and their incorporation into lipid bilayers, the macromolecular complex stays mostly intact except for CaM which can dissociate from the RyR2 complex in minutes.Heart failure is a complex disorder involving changes in Ca2+ handling protein expression, Ca2+ homeostasis and remodelling of the RyR2 complex. We compare RyR2 activity from healthy and failing human hearts and study their regulation by Ca2+ in the presence and absence of CaM. RyR2 was isolated from healthy human hearts and hearts with ischemic cardiomyopathy with ethics approval (H-2009-0369) and incorporated into lipid bilayers. CaM (0.5 μM with 0.1 uM Ca2+) caused a reduction in RyR2 Po of 57% ± 6% from failing human heart but did not affect RyRs from healthy hearts. Thus, action of CaM in the RyR complex is influenced by its remodelling that occurs in the failing heart.

Physiological functions and regulation of TRPC channels.

The TRP-canonical (TRPC) subfamily, which consists of seven members (TRPC1-TRPC7), are Ca(2+)-permeable cation channels that are activated in response to receptor-mediated PIP2 hydrolysis via store-dependent and store-independent mechanisms. These channels are involved in a variety of physiological functions in different cell types and tissues. Of these, TRPC6 has been linked to a channelopathy resulting in human disease. Two key players of the store-dependent regulatory pathway, STIM1 and Orai1, interact with some TRPC channels to gate and regulate channel activity. The Ca(2+) influx mediated by TRPC channels generates distinct intracellular Ca(2+) signals that regulate downstream signaling events and consequent cell functions. This requires localization of TRPC channels in specific plasma membrane microdomains and precise regulation of channel function which is coordinated by various scaffolding, trafficking, and regulatory proteins.

Electrophysiological Characterization of the Activity and Regulation of the Mitochondrial Calcium Uniporter

Mitochondrial calcium uptake is present in all eukaryotic living organisms and represents a critical point because it is implicated in highly sophisticated processes that have multiple consequences for the cells' survival or death.Ca2+ uptake by energized isolated mitochondria was directly measured for the first time half a century ago (Vasington and Murphy, 1962), but the molecular identity of the pore-forming subunit of the Ca2+ mitochondrial uptake protein complex MCU (mitochondrial calcium uniport), became available only in 2011, thank to the combination of bioinformatics, cell biology and the application of planar lipid bilayers experiments using recombinant MCU protein (De Stefani et al., 2011). Today, it is clear that the channel responsible for Ca2+ uptake is an inner mitochondrial membrane protein that works as a highly selective channel which can be inhibited by ruthenium red and gadolinium (Kirichok et al., 2004, De Stefani et al., 2011).The application of electrophysiology in addition to cell biology techniques opens new perspectives to elucidate the physiological and pathological relevance of MCU and to directly study the role of numerous putative regulators/additional components of the MCU. For example, recent evidence shows that MCU is able to form hetero-oligomers with a protein, called MCUb, which acts as a dominant-negative pore forming subunit (Raffaello et al., 2013). Here we report further biophysical characterization of recombinant MCU studied in BLM experiments where possible regulatory factors are not present. Furthermore, we describe our studies using molecules, shown to affect calcium uptake in intact cells, like MICU1 (Perocchi et al., 2013). Given that regulation of MCU activity in intact cells by MICU1 however is controversial (Mallilankaraman et al., 2012a/b; Csordas et al., 2013), our results provide an important, direct evidence in favor of direct regulation of channel activity.

CLCNKB mutations causing mild Bartter syndrome profoundly alter the pH and Ca2+ dependence of ClC-Kb channels

ClC-Kb, a member of the ClC family of Cl(-) channels/transporters, plays a major role in the absorption of NaCl in the distal nephron. CLCNKB mutations cause Bartter syndrome type 3, a hereditary renal salt-wasting tubulopathy. Here, we investigate the functional consequences of a Val to Met substitution at position 170 (V170M, α helix F), which was detected in eight patients displaying a mild phenotype. Conductance and surface expression were reduced by ~40-50%. The regulation of channel activity by external H(+) and Ca(2+) is a characteristic property of ClC-Kb. Inhibition by external H(+) was dramatically altered, with pKH shifting from 7.6 to 6.0. Stimulation by external Ca(2+) on the other hand was no longer detectable at pH 7.4, but was still present at acidic pH values. Functionally, these regulatory modifications partly counterbalance the reduced surface expression by rendering V170M hyperactive. Pathogenic Met170 seems to interact with another methionine on α helix H (Met227) since diverse mutations at this site partly removed pH sensitivity alterations of V170M ClC-Kb. Exploring other disease-associated mutations, we found that a Pro to Leu substitution at position 124 (α helix D, Simon et al., Nat Genet 1997, 17:171-178) had functional consequences similar to those of V170M. In conclusion, we report here for the first time that ClC-Kb disease-causing mutations located around the selectivity filter can result in both reduced surface expression and hyperactivity in heterologous expression systems. This interplay must be considered when analyzing the mild phenotype of patients with type 3 Bartter syndrome.

Tetraspanin-13 modulates voltage-gated CaV2.2 Ca2+ channels

Integration of voltage-gated Ca2+ channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca2+ channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the α1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca2+ channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba2+ current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca2+ channel activity in defined synaptic membrane compartments and thereby influences transmitter release.

ENaC Regulation by Proteases and Shear Stress

Epithelial Na(+) channels (ENaCs) are comprised of subunits that have large extracellular regions linked to membrane spanning domains where the channel pore and gate reside. A variety of external factors modify channel activity by interacting at sites within extracellular regions that lead to conformational changes that are transmitted to the channel gate and alter channel open probability. Our review addresses two external factors that have important roles in regulating channel activity, proteases and laminar shear stress.

Characterization of the first honeybee Ca2+ channel subunit reveals two novel species- and splicing-specific modes of regulation of channel inactivation

The honeybee is a model system to study learning and memory, and Ca(2+) signals play a key role in these processes. We have cloned, expressed, and characterized the first honeybee Ca(2+) channel subunit. We identified two splice variants of the Apis CaVβ Ca(2+) channel subunit (Am-CaVβ) and demonstrated expression in muscle and neurons. Although AmCaVβ shares with vertebrate CaVβ subunits the SH3 and GK domains, it beholds a unique N terminus that is alternatively spliced in the first exon to produce a long (a) and short (b) variant. When expressed with the CaV2 channels both, AmCaVβa and AmCaVβb, increase current amplitude, shift the voltage-sensitivity of the channel, and slow channel inactivation as the vertebrate CaVβ2a subunit does. However, as opposed to CaVβ2a, slow inactivation induced by Am-CaVβa was insensitive to palmitoylation but displayed a unique PI3K sensitivity. Inactivation produced by the b variant was PI3K-insensitive but staurosporine/H89-sensitive. Deletion of the first exon suppressed the sensitivity to PI3K inhibitors, staurosporine, or H89. Recording of Ba(2+) currents in Apis neurons or muscle cells evidenced a sensitivity to PI3K inhibitors and H89, suggesting that both AmCaVβ variants may be important to couple cell signaling to Ca(2+) entry in vivo. Functional interactions with phospho-inositide and identification of phosphorylation sites in AmCaVβa and AmCaVβb N termini, respectively, suggest that AmCaVβ splicing promoted two novel and alternative modes of regulation of channel activity with specific signaling pathways. This is the first description of a splicing-dependent kinase switch in the regulation of Ca(2+) channel activity by CaVβ subunit.

Triple Cysteine Module within M-Type K+Channels Mediates Reciprocal Channel Modulation by Nitric Oxide and Reactive Oxygen Species

We have identified a new signaling role for nitric oxide (NO) in neurons from the trigeminal ganglia (TG). We show that in rat sensory neurons from the TG the NO donor, S-nitroso-N-acetyl-dl-penicillamine, inhibited M-current. This inhibitory effect was blocked by NO scavenging, while inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. Moreover NO increased neuronal excitability and calcitonin gene-related peptide (CGRP) release and these effects could be prevented by perturbing M-channel function. First, NO-induced depolarization was prevented by pre-application of the M-channel blocker XE991 and second, NO-induced increase in CGRP release was prevented by incubation with the M-channel opener retigabine. We investigated the mechanism of the effects of NO on M-channels and identified a site of action of NO to be the redox modulatory site at the triplet of cysteines within the cytosolic linker between transmembrane domains 2 and 3, which is also a site of oxidative modification of M-channels by reactive oxygen species (ROS). NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert fine control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels, which mediates reciprocal regulation of channel activity by NO and ROS. This sensor may play an important role in mediating excitatory effects of NO in such trigeminal disorders as headache and migraine.

Targeting the high‐conductance Ca2+‐activated K+ (BK) channel as vasodilator therapy for pulmonary hypertension

Pulmonary arterial hypertension (PAH) is characterized by a loss of nitric oxide, prostacyclin, and other vasodilator molecules resulting in pulmonary vasoconstriction. In contrast, our Western blot studies detected a 4.9‐fold increase in expression of the high‐conductance Ca2+‐activated K+ (BK) channel in the pulmonary arteries (PA) of rats with hypoxia‐induced PAH (n=6), suggesting the upregulation of a potent vasodilator influence. However, it is possible that BK channels are inactivated in hypoxia‐induced PAH, since RT‐PCR confirmed that the BK α‐subunit in rat PA contains a hypoxia‐sensitive STREX region known to negatively regulate channel activity. Considering these factors, we hypothesized that pharmacological activation of over‐expressed BK channels in the VSMCs of affected PA may enact vasodilation as a therapy for PAH. Patch‐clamp studies showed that NS11021 (1, 3 and 10 μmol/L), a novel pharmacological BK channel opener, increased whole‐cell K+ current by 1.9‐fold in VSMCs of PA from control rats (n=3). Similar concentrations of NS11021 also partially reversed hypoxia‐induced constrictions of rat PA rings in tension‐recording studies (n=3). These results suggest that BK channel openers represent a potential vasodilator therapy for PAH, a hypothesis we plan to test using pulmonary biotelemetry in rat models of PAH. Supported by UL1RR029884 (NDD) from the NIH.

Specific phosphorylation sites underlie the stimulation of a large conductance, Ca2+‐activated K+channel by cGMP‐dependent protein kinase

Smooth muscle contractility and neuronal excitability are regulated by large conductance, Ca(2+)-activated K(+) (BKCa) channels, the activity of which can be increased after modulation by type I cGMP-dependent protein kinase (cGKI) via nitric oxide (NO)/cGMP signaling. Our study focused on identifying key phosphorylation sites within the BKCa channel underlying functional enhancement of channel activity by cGKI. BKCa channel phosphorylation by cGKIα was characterized biochemically using radiolabeled ATP, and regulation of channel activity by NO/cGMP signaling was quantified in rat aortic A7r5 smooth muscle cells by cell-attached patch-clamp recording. Serine to alanine substitutions at 3 of 6 putative cGKI phosphorylation sites (Ser691, Ser873, and Ser1112) in the BKCa α subunit individually reduced direct channel phosphorylation by 25-60% and blocked BKCa activation by either an NO donor or a membrane-permeable cGMP by 80-100%. Acute inhibition of cGKI prevented stimulus-evoked enhancement of BKCa channel activity. Our data further suggest that augmentation of BKCa activity by NO/cGMP/cGKI signaling requires phosphorylation at all 3 sites and is independent of elevations in [Ca(2+)]i. Phosphorylation of 3 specific Ser residues within the murine BKCa α subunit by cGKIα accounts for the enhanced BKCa channel activity induced by elevated [cGMP]i in situ.

Regulation of large conductance calcium- and voltage-activated potassium (BK) channels by S-palmitoylation

BK (large conductance calcium- and voltage-activated potassium) channels are important determinants of physiological control in the nervous, endocrine and vascular systems with channel dysfunction associated with major disorders ranging from epilepsy to hypertension and obesity. Thus the mechanisms that control channel surface expression and/or activity are important determinants of their (patho)physiological function. BK channels are S-acylated (palmitoylated) at two distinct sites within the N- and C-terminus of the pore-forming α-subunit. Palmitoylation of the N-terminus controls channel trafficking and surface expression whereas palmitoylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. Recent studies are beginning to reveal mechanistic insights into how palmitoylation controls channel trafficking and cross-talk with phosphorylation-dependent signalling pathways. Intriguingly, each site of palmitoylation is regulated by distinct zDHHCs (palmitoyl acyltransferases) and APTs (acyl thioesterases). This supports that different mechanisms may control substrate specificity by zDHHCs and APTs even within the same target protein. As palmitoylation is dynamically regulated, this fundamental post-translational modification represents an important determinant of BK channel physiology in health and disease.

M-Type K+ Channel as Plasma Membrane Nitric Oxide and Reactive Oxygen Species Sensor

M-type K+ channels control excitability of many neuron types. M channel activity is potently enhanced by oxidative modification of triple cysteine pocket within the cytosolic linker between transmembrane domains 2 and 3. Thus, in sensory neurons from dorsal root (DRG) and trigeminal (TG) ganglia M channel activity is increased by the mitochondrial release of reactive oxygen species (ROS) induced by neuromodulator substance P. Here we identified a new signalling role for nitric oxide (NO) in TG sensory neurons. We show that in rat TG neurons, the NO donor, S-Nitroso-N-acetyl-DL-penicillamine (SNAP), inhibited M-current by 53±12% (n=12). This inhibitory effect was blocked by scavenging of NO and inhibition of NO synthases increased M-current, suggesting that tonic NO levels inhibit M-current in TG neurons. NO increased release of calcitonin gene-related peptide (CGRP) from TG neurons (130±10% of control, p≤0.05), consistent with an increase in neuronal excitability. Importantly, incubation with the M-channel opener retigabine completely abolished increases in CGRP release. We further investigated the mechanism of the effects of NO on M channels and identified a site of action of NO to be the same triplet of cysteines, which is also a site of oxidative modification of M channels by ROS. We now show that the same triplet of cysteines can be S-nitrosylated in the presence of SNAP. We show that NO and oxidative modifications have opposing effects on M-current, suggesting that a tightly controlled local redox and NO environment will exert control over M-channel activity and thus neuronal excitability. Together our data have identified a dynamic redox sensor within neuronal M-channels which mediates reciprocal regulation of channel activity by S-nitrosylation and oxidative modification. This sensor may play an important role in controlling neuronal excitability by redox and NO-related mechanisms.

SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity

BackgroundTransient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions.ResultsHere we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface.ConclusionSNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.

Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished. Collectively, these results rule out Ca2+ regulation of channel activity by direct binding to the luminal aspect of the channel.

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

Differential Regulation by Cyclic Nucleotides of the CNGA4 and CNGB1b Subunits in Olfactory Cyclic Nucleotide–Gated Channels

Olfactory cyclic nucleotide-gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide-binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.

A peptide uncoupling CRMP-2 from the presynaptic Ca2+ channel complex demonstrates efficacy in animal models of migraine and AIDS therapy-induced neuropathy

Biological, genetic, and clinical data provide compelling proof for N-type voltage-gated calcium channels (CaV2.2) as therapeutic targets for chronic pain. While decreasing channel function is ultimately anti-nociceptive, directly targeting the channel can lead to multiple adverse effects. Targeting regulators of channel activity may facilitate improved analgesic properties associated with channel block and afford a broader therapeutic window. Towards this end, we recently identified a short peptide, designated CBD3, derived from collapsin response mediator protein 2 (CRMP-2) that suppressed inflammatory and neuropathic hypersensitivity by inhibiting CRMP-2 binding to CaV2.2 [Brittain et al., Nature Medicine 17:822-829 (2011)]. Rodents administered CBD3 intraperitoneally, fused to the HIV TAT protein cell penetrating domain, exhibited antinociception lasting ~4 hours highlighting potential instability, limited oral bioavailability, and/or rapid elimination of peptide. This report focuses on improving upon the parental CBD3 peptide. Using SPOTScan analysis of synthetic versions of the parental CBD3 peptide, we identified peptides harboring single amino acid mutations that bound with greater affinity to CaV2.2. One such peptide, harboring a phenylalanine instead of glycine (G14F), was tested in rodent models of migraine and neuropathic pain. In vivo laser Doppler blood flowmetry measure of capsaicin-induced meningeal vascular responses related to headache pain was almost completely suppressed by dural application of the G14F peptide. The G14F mutant peptide, administered intraperitoneally, also exhibited greater antinociception in Stavudine (2'-3'-didehydro-2'-3'-dideoxythymidine (d4T)/Zerit®) model of AIDS therapy-induced peripheral neuropathy compared to the parent CBD3 peptide. These results demonstrate the patent translational value of small biologic drugs targeting CaV2.2 for management of clinical pain.

Multiple Ca2+ Binding Sites in the RCK Domain Contribute to Gating in the MthK K+ Channel

MthK is a prokaryotic Ca2+-gated K+ channel that has yielded structural insight toward mechanisms of RCK domain controlled channel gating. In MthK, a “gating ring” of eight RCK domains regulates channel activation that is steeply dependent on [Ca2+], with a Hill coefficient >8. Using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca2+ binding sites, two of which are located at the interfaces between adjacent RCK domains. Charge-neutralizing mutations of individual Ca2+-coordinating sidechains at each of the three sites decrease Ca2+-sensitivity of the channel, shifting the EC50 for Ca2+-activation by up to 8.4-fold. Combined mutations at pairs of sites decreased the Ca2+-sensitivity of mutant channels in an approximately energetically additive manner, suggesting that Ca2+ binding at each site may act independent of one another to faciliate channel opening. Finally, a combined mutation at all three sites effectively eliminates Ca2+-dependent activation of the channel through very high [Ca2+]. The additional Ca2+ binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity, and crystal structures of the MthK RCK domain in the presence of Ca2+ suggest points at which subunits may physically interact to facilitate channel opening.