Reduced Channel Open Probability Research Articles

Loss of CaV1.3 RNA editing enhances mouse hippocampal plasticity, learning, and memory

L-type CaV1.3 calcium channels are expressed on the dendrites and soma of neurons, and there is a paucity of information about its role in hippocampal plasticity. Here, by genetic targeting to ablate CaV1.3 RNA editing, we demonstrate that unedited CaV1.3ΔECS mice exhibited improved learning and enhanced long-term memory, supporting a functional role of RNA editing in behavior. Significantly, the editing paradox that functional recoding of CaV1.3 RNA editing sites slows Ca2+-dependent inactivation to increase Ca2+ influx but reduces channel open probability to decrease Ca2+ influx was resolved. Mechanistically, using hippocampal slice recordings, we provide evidence that unedited CaV1.3 channels permitted larger Ca2+ influx into the hippocampal pyramidal neurons to bolster neuronal excitability, synaptic transmission, late long-term potentiation, and increased dendritic arborization. Of note, RNA editing of the CaV1.3 IQ-domain was found to be evolutionarily conserved in mammals, which lends support to the importance of the functional recoding of the CaV1.3 channel in brain function.

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail.

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits.

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, β, and γ). We previously reported that cytoplasmic residues on the β (βCys-43 and βCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over β subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking β subunit palmitoylation sites. β subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking β and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

The mammalian skeletal muscle DHPR has larger Ca2+ conductance and is phylogenetically ancient to the early ray-finned fish sterlet (Acipenser ruthenus)

The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po.

Disruption of S2-M4 linker coupling reveals novel subunit-specific contributions to N-methyl-d-aspartate receptor function and ethanol sensitivity

The N-methyl-d-aspartate (NMDA) receptor is a glutamatergic ion channel and is a known site of ethanol action. Evidence suggests that ethanol inhibits NMDA receptor activity by reducing channel open probability and mean open time potentially via interaction with specific residues within the transmembrane (M) domains 3 and 4 of GluN subunits. Recent models of NMDAR function demonstrate that extracellular residues near the M domains are key regulators of gating, suggesting that they may contribute to ethanol sensitivity. To test this, we substituted cysteines at key positions in GluN1 and GluN2 M3-S2 and S2-M4 regions previously shown to affect channel open probability and mean open time similar to ethanol treatment. Although crosslinking of these domains was predicted to restrict linker domain movement and occlude ethanol inhibition, only intra-GluN1 M1:M4 linker-crosslinked receptors showed a decrease in ethanol sensitivity. For the converse experiment, we expressed NMDARs with glycine substitutions in the S2-M4 domain of GluN subunits to enhance M4 flexibility and recorded currents in response to ethanol. Glycine substitution in the GluN1 S2-M4 region significantly decreased glutamate potency of GluN1(A804G)/GluN2A receptors, while GluN1(A804G)/GluN2B receptors exhibited no change in glutamate sensitivity. In contrast, GluN1/GluN2B(S811G) receptors showed a 10-fold increase in glutamate potency while GluN1/GluN2A(S810G) receptors showed no change. Surprisingly, while S2-M4 glycine substitutions modulated ethanol sensitivity, this was observed only in receptors that did not display a change in agonist potency. Overall, these results suggest that S2-M4 linkers strongly influence receptor function and modestly impact ethanol efficacy in a subunit- and receptor subtype-dependent manner.

Pendrin gene ablation alters ENaC subcellular distribution and open probability.

The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability.

Compromised Salt Dependence of ENaC Activity Upon Deletion of AT1 Receptors

ENaC-mediated sodium reabsorption in the collecting duct (CD) is an essential determinant of blood pressure in humans. Cumulative evidence suggests direct action of Angiotensin II (Ang II) on ENaC activity in the CD, independently of the classical aldosterone signaling. The significance of regulation by Ang II becomes particularly important during Ang II dependent hypertension, when overactive ENaC is poorly regulated via aldosterone – mineralocorticoid receptor (MR) axis. Stimulatory Ang II actions on ENaC are mediated by AT1 receptors (AT1R). AT1R knockouts have decreased blood pressure despite elevated aldosterone levels. This indicates that aldosterone-MR cascade alone cannot provide sufficient input to adequately regulate ENaC-dependent sodium re-uptake. Using freshly isolated split-opened CDs we found that genetic deletion of AT1R markedly diminishes ENaC activity on low and regular sodium intake, significantly blunting salt-dependent regulation of ENaC. The observed decrease of ENaC activity was mainly attributed to a reduced channel open probability (Po). Pharmacological inhibition of AT1R with losartan in wild-type animals reproduces the effect of genetic ablation of AT1R on ENaC activity. Interestingly, saturation of aldosterone signaling with DOCA has minimal stimulatory effect on ENaC upon AT1R deletion, which can be explained by a markedly diminished MR abundance in the CD cells of the knockouts. Overall, genetic ablation of AR1R elicits a complex inhibitory effect on ENaC activity, directly attenuating ENaC Po and precluding adequate compensation by aldosterone cascade due to decreased MR expression.

Cysteine Palmitoylation of the γ Subunit Has a Dominant Role in Modulating Activity of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na(+) self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na(+) self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating.

Two serine residues on GluN2A C-terminal tails control NMDA receptor current decay times

NMDA receptors are glutamate-activated, Ca2+-permeable ion channels with critical roles in synaptic transmission and plasticity. The shape and size of their current is modulated by several kinase/phosphatase systems, and numerous residues located on the receptors’ intracellular C-termini are phosphorylated in vivo. To investigate the mechanisms by which phosphorylation may control channel gating, we examined the single-channel behaviors of receptors carrying the S900A or S929A substitution in their GluN2A subunits and thus were rendered resistant to phosphorylation at those sites. We found that the mutations reduced channel open probability primarily by increasing the frequency of desensitized events. The kinetic models we developed revealed complex but similar changes in mechanism for the two mutants, leading to the view that dephosphorylation at either site may cause receptors to activate slower, deactivate faster and desensitize more frequently. This modulatory mechanism is consistent with the proposed roles for these residues in Ca2+-dependent desensitization and calcineurin-mediated reduction of current during brain development.

Sphingosine and FTY720 are potent inhibitors of the transient receptor potential melastatin 7 (TRPM7) channels

Transient receptor potential melastatin 7 (TRPM7) is a unique channel kinase which is crucial for various physiological functions. However, the mechanism by which TRPM7 is gated and modulated is not fully understood. To better understand how modulation of TRPM7 may impact biological processes, we investigated if TRPM7 can be regulated by the phospholipids sphingosine (SPH) and sphingosine-1-phosphate (S1P), two potent bioactive sphingolipids that mediate a variety of physiological functions. Moreover, we also tested the effects of the structural analogues of SPH, N,N-dimethyl-D-erythro-sphingosine (DMS), ceramides and FTY720 on TRPM7. HEK293 cells stably expressing TRPM7 were used for whole-cell, single-channel and macropatch current recordings. Cardiac fibroblasts were used for native TRPM7 current recording. SPH potently inhibited TRPM7 in a concentration-dependent manner, whereas S1P and other ceramides did not produce noticeable effects. DMS also markedly inhibited TRPM7. Moreover, FTY720, an immunosuppressant and the first oral drug for treatment of multiple sclerosis, inhibited TRPM7 with a similar potency to that of SPH. In contrast, FTY720-P has no effect on TRPM7. It appears that SPH and FTY720 inhibit TRPM7 by reducing channel open probability. Furthermore, endogenous TRPM7 in cardiac fibroblasts was markedly inhibited by SPH, DMS and FTY720. This is the first study demonstrating that SPH and FTY720 are potent inhibitors of TRPM7. Our results not only provide a new modulation mechanism of TRPM7, but also suggest that TRPM7 may serve as a direct target of SPH and FTY720, thereby mediating S1P-independent physiological/pathological functions of SPH and FTY720.

Calcium-Flux-Mediated Modulation of InsP3R Channel Activity by ER Luminal Calcium Concentration

The ubiquitous, mostly endoplasmic reticulum (ER)-localized InsP3R channel modulates cytoplasmic free [Ca2+] ([Ca2+]i) to generate complex Ca2+ signals by releasing Ca2+ stored in the ER lumen. Whereas cytoplasmic InsP3 activation and biphasic [Ca2+]i regulation of the channel have been well studied, its regulation by ER luminal free [Ca2+] ([Ca2+]ER) is poorly understood and controversial. Here, we used excised luminal-side-out nuclear patch clamping with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R (InsP3R-3) channel activity. In optimal 2 μM [Ca2+]i (buffered by 0.5 mM diBrBAPTA) and sub-saturating 3 μM InsP3, jumps of [Ca2+]ER from 70 nM (no Ca2+ flux through channel) to 300 μM (substantial Ca2+ flux) reduced channel open probability (Po) significantly. This inhibition was abrogated when saturating 10 μM InsP3 was used, but was restored when [Ca2+]ER was raised to 2 mM. This [Ca2+]ER effect exhibited a biphasic dependence on [Ca2+]i because jumps of [Ca2+]ER (70 nM to 300 μM) in 3 μM InsP3 increased Po in sub-optimal 70 nM Ca2+i. These effects of [Ca2+]ER were attenuated when an electrical potential was applied to oppose Ca2+ flux through the channel. Importantly, the effects of [Ca2+]ER on channel activity depended on cytoplasmic Ca2+ buffering conditions: it was stronger when [Ca2+]i was weakly buffered by 0.1 mM HEDTA (a slow Ca2+ chelator) but was completely abolished with 5 mM diBrBAPTA buffering. These observations are completely accounted for by Ca2+ flux driven by [Ca2+]ER through the open InsP3R channel raising local [Ca2+]i around the channel to regulate its Po through its cytoplasmic activating and inhibitory Ca2+-binding sites. These results provide no evidence for a role of [Ca2+]ER in regulating InsP3R-3 activity by effects mediated directly on the luminal aspect of the channel.

Zinc Effects on NMDA Receptor Gating Kinetics

Zinc accumulates in the synaptic vesicles of certain glutamatergic forebrain neurons and modulates neuronal excitability and synaptic plasticity by multiple poorly understood mechanisms. Zinc directly inhibits NMDA-sensitive glutamate-gated channels by two separate mechanisms: high-affinity binding to N-terminal domains of GluN2A subunits reduces channel open probability, and low-affinity voltage-dependent binding to pore-lining residues blocks the channel. Insight into the high-affinity allosteric effect has been hampered by the receptor's complex gating; multiple, sometimes coupled, modulatory mechanisms; and practical difficulties in avoiding transient block by residual Mg 2+. To sidestep these challenges, we examined how nanomolar zinc concentrations changed the gating kinetics of individual block-resistant receptors. We found that block-insensitive channels had lower intrinsic open probabilities but retained high sensitivity to zinc inhibition. Binding of zinc to these receptors resulted in longer closures and shorter openings within bursts of activity but had no effect on interburst intervals. Based on kinetic modeling of these data, we conclude that zinc-bound receptors have higher energy barriers to opening and less stable open states. We tested this model for its ability to predict zinc-dependent changes in macroscopic responses and to infer the impact of nanomolar zinc concentrations on synaptic currents mediated by 2A-type NMDA receptors.

Bradykinin acutely inhibits activity of the epithelial Na+channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).

Interaction with caveolin-1 modulates vascular ATP-sensitive potassium (KATP) channel activity

ATP-sensitive potassium channels (K(ATP) channels) of arterial smooth muscle are important regulators of arterial tone, and hence blood flow, in response to vasoactive transmitters. Recent biochemical and electron microscopic evidence suggests that these channels localise to small vesicular invaginations of the plasma membrane, known as caveolae, and interact with the caveolae-associated protein, caveolin. Here we report that interaction with caveolin functionally regulates the activity of the vascular subtype of K(ATP) channel, Kir6.1/SUR2B. Pinacidil-evoked recombinant whole-cell Kir6.1/SUR2B currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 +/- 8.3 pA pF(1), n = 8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 +/- 35.9 pA pF(1), n = 6; P < 0.05) indicating that interaction with caveolin may inhibit channel activity. Inclusion in the pipette-filling solution of a peptide corresponding to the scaffolding domain of caveolin-1 had a similar inhibitory effect on whole-cell Kir6.1/SUR2B currents as co-expression with full-length caveolin-1, while a scrambled version of the same peptide had no effect. Interestingly, intracellular dialysis of vascular smooth muscle cells with the caveolin-1 scaffolding domain peptide (SDP) also caused inhibition of pinacidil-evoked native whole-cell K(ATP) currents, indicating that a significant proportion of vascular K(ATP) channels are susceptible to block by exogenously applied SDP. In cell-attached recordings of Kir6.1/SUR2B single channel activity, the presence of caveolin-1 significantly reduced channel open probability (from 0.05 +/- 0.01 to 0.005 +/- 0.001; P < 0.05) and the amount of time spent in a relatively long-lived open state. These changes in kinetic behaviour can be explained by a caveolin-induced shift in the channel's sensitivity to its physiological regulator MgADP. Our findings thus suggest that interaction with caveolin-1 suppresses vascular-type K(ATP) channel activity. Since caveolin expression is regulated by cellular free cholesterol and plasma levels of low-density lipoprotein (LDL), this interaction may have implications in both the physiological and pathophysiological control of vascular function.

Mechanism of verapamil action on wild‐type and slow‐channel mutant human muscle acetylcholine receptor

Verapamil, a Ca(2+) channel blocker widely used in clinical practice, also affects the properties of frog and mouse muscle acetylcholine receptor (AChR). Here, we examine the mechanism of action of verapamil on human wild-type and slow-channel mutant muscle AChRs harboring in any subunit a valine-to-alanine mutation of 13' residue of the pore-lining M2 transmembrane segment. Verapamil, after a pre-treatment of 0.5-10 s, accelerated the decay of whole-cell or macroscopic outside-out currents within milliseconds of ACh application even at clinically attainable doses. Recordings of unitary events in the cell-attached and outside-out configurations showed that verapamil does not alter single-channel conductance, but reduces channel open probability, by prolonging the dwell time into the closed state for wild-type and all mutant AChR. The duration of channel openings decreased only for the epsilonV265A-AChR, by shortening the longest exponential component of the open-time distribution. These results provide a rationale for the therapeutic use of verapamil in the slow-channel syndrome and emphasize the major role played by epsilon subunit in controlling the functional properties of human muscle AChR, as revealed by the peculiar alterations imparted by mutations in this subunit.

Caveolin-1 Inhibits Vascular KATP Channels by Modulating Channel Sensitivity to MgADP

Caveolae are small vesicular invaginations of the plasma membrane enriched in cholesterol, sphingolipids and the cholesterol-binding protein, caveolin [1]. Aside from roles in caveolae formation and stability, caveolins interact with many caveolae-localised molecules via an N-terminal region known as the caveolin scaffolding domain. One of the many signalling proteins that reside in caveolae and interact with caveolins is the vascular ATP-sensitive potassium (KATP) channel [2], an ion channel important in the control of smooth muscle contractility and so blood flow. Here we investigate the potential role of caveolin in regulating KATP channel behaviour. Pinacidil-evoked recombinant whole-cell KATP (Kir6.1/SUR2B) currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 ± 8.3pA/pF, n=8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 ± 35.9pA/pF, n=6; p<0.05), indicating that interaction with caveolin-1 may inhibit channel activity. The addition of a peptide corresponding to the caveolin-1 scaffolding domain to the pipette-filling solution had a similar inhibitory effect on whole-cell recombinant KATP currents. In cell-attached patch clamp recordings, the presence of caveolin-1 significantly reduced channel open probability and the amount of time spent in a relatively long-lived open state. Sensitivity of the channel to its physiological regulator MgADP was significantly altered by caveolin-1 and can explain these changes in channel kinetic behaviour. Our findings suggest that interaction with caveolin-1 has an inhibitory effect on arterial-type KATP channel activity that may be important in both the physiological and pathophysiological control of vascular function. 1. Parton & Simons (2007). Nat Rev Mol Cell Biol8, 185-194. 2. Sampson et al. (2004). Circ Res95, 1012-1018. Supported by the BBSRC and the BHF

Phosphatidylinositol 4,5-bisphosphate and loss of PLCγ activity inhibit TRPM channels required for oscillatory Ca2+ signaling

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) oscillations that control muscle contractions required for defecation. Two highly Ca(2+)-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLCgamma in a common signaling pathway that regulates IP(3)-dependent intracellular Ca(2+) release. A second PLC, PLCbeta, is also required for IP(3)-dependent Ca(2+) oscillations, but functions in an independent signaling mechanism. PLCgamma generates IP(3) that regulates IP(3) receptor activity. We demonstrate here that PLCgamma via hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) also regulates GON-2/GTL-1 function. Knockdown of PLCgamma but not PLCbeta activity by RNA interference (RNAi) inhibits channel activity approximately 80%. Inhibition is fully reversed by agents that deplete PIP(2) levels. PIP(2) added to the patch pipette has no effect on channel activity in PLCgamma RNAi cells. However, in control cells, 10 microM PIP(2) inhibits whole cell current approximately 80%. Channel inhibition by phospholipids is selective for PIP(2) with an IC(50) value of 2.6 microM. Elevated PIP(2) levels have no effect on channel voltage and Ca(2+) sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of PIP(2) by PLCgamma functions in the activation of both the IP(3) receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell Ca(2+) influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate IP(3) receptor function. PIP(2)-dependent regulation of GON-2/GTL-1 may provide a mechanism to coordinate plasma membrane Ca(2+) influx with PLCgamma and IP(3) receptor activity as well as intracellular Ca(2+) store depletion.

Depolarization-induced Potentiation Of Cav1.1 Does Not Require The Distal C-terminus

In adult skeletal muscle, the majority of the L-type Ca2+ channel CaV1.1 subunit is truncated post-translationally at residue 1664 (PNAS, 102:5274-79), raising the question of the functional role of the distal residues (1665-1873). It has been suggested (J Neurosci.17:1243-55; J Biol Chem. 277:4079-87) that (i) the distal C-terminus is non-covalently associated with the remainder of the channel, (ii) reduces channel open probability, and (iii) loses this inhibitory effect as a result of phosphorylation during strong depolarization. In regard to point (ii), previous analysis of L-type ionic conductance (G) and membrane gating charge movements (Q) demonstrated that channel open probability (i.e., G/Q ratio) was indistinguishable for full-length or truncated (at 1662) CaV1.1 expressed in dysgenic myotubes (Nature 360:169-171). Here we have investigated the effects of removing the distal C-terminus on depolarization-induced potentiation of CaV1.1. Specifically, tail currents were measured for repolarization to -30 mV following a 200 ms depolarization to either +40 or +90 mV. For both full-length and truncated CaV1.1, tail currents were both larger (∼2.5-fold) and more slowly decaying (∼2-fold) following the +90 mV depolarization. Thus, we find no evidence for a role of the CaV1.1 distal C-terminus in depolarization-induced potentiation.We are currently examining the role of the CaV1.2 distal C-terminus by co-expression of full-length or truncated (1669) CaV1.2 in tsA-201 cells together with β2a and α2δ1. In agreement with previous work (J Physiol. 576:87-102, and in contrast to CaV1.1, truncation of CaV1.2 resulted in ∼4-fold increase in the G/Q ratio. We are currently investigating the ability of the truncated CaV1.2 to undergo depolarization-induced potentiation. Supported by NIH (NS24444) and MDA grants to KGB.

AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na+ across the lung epithelium via amiloride-sensitive Na+ channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na+ conductance (GNa+) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to GNa+: a 5-pS highly Na+ selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to −38 mV and 0 mV to −35 mV. Amiloride at 1 μM inhibited HSC activity and 56% of short-circuit current (Isc), whereas 10 μM amiloride partially reduced NSC activity and inhibited a further 30% of Isc. Neither conductance was associated with CNG channels as there was no effect of 10 μM pimoside on Isc, HSC, or NSC activity, and 8-bromo-cGMP (0.3–0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na+-permeable channels by a mechanism that likely reduces channel open probability.