Regulation Of Channel Function Research Articles

Calmodulin binding to M‐type K+ channels assayed by TIRF/FRET in living cells

Calmodulin (CaM) binds to KCNQ2-4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca2+ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2-4 carboxy termini and CaM proteins to determine the Ca2+ dependence of the interaction in vitro. The assays showed substantial Ca2+ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM-channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2-4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca2+]i, low [Ca2+]i or high [Ca2+]i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca2+ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2-4 channels and DN CaM was robust, and not significantly Ca2+ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2-4 channels and CaM are likely to have Ca2+-dependent and Ca2+-independent components.

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.

Abstract 637: A Pharmacogenetic Screen for Modifiers of Drug Induced QT Prolongation Reveals 15 Novel Genes.

Introduction: Variability in response to therapeutic drugs is a vexing problem that has recently begun to yield to intense investigatory efforts. Rigorous pharmacogenetic studies will require model systems that recapitulate the complexity of individual drug responses. Methods: We have employed the voltage sensitive fluorescent dye di 4-ANEPPS and high speed CCD camera acquisition to study cardiac repolarization in embryonic zebrafish. We used a known zebrafish KCNH2 mutant, anemone toxin II (ATXII) and dofetilide to perturb cardiac repolarization. We also undertook a screen of a known panel of 294 mutant zebrafish lines to identify genes that modify the response to IKr block. Results: Using optical mapping, we demonstrate action potential prolongation (Figure 1a,b ), reduced repolarization reserve (Figure 1d ), and spontaneous early afterdepolarizations in zebrafish harboring a mutation in KCNH2. We also show that augmentation of late sodium current using anemone toxin II prolongs action potential duration (Figure 1c ). Using this model we have conducted a pharmacogenetic screen for modifiers of drug-induced QT prolongation, and have identified 15 genes that modulate the response to IKr block, including three members of the heg-san-vtn pathway. Conclusion: Together these data implicate a complex network of non-ion channel proteins functioning in membrane microdomains to regulate channel function, and validate high-throughput screening in zebrafish to dissect complex biologic systems.

Regulation of TRP channels by N-linked glycosylation

A subset of TRP channel proteins undergoes regulatory N-linked glycosylation. A glycosylation site in the first extracellular loop of TRPV5 is enzymatically cleaved by a secreted glucuronidase, indirectly regulating channel function. Members of the TRPC family share a similar site, although details about a regulatory role are lacking. A second conserved TRP channel glycosylation site is found immediately adjacent to the channel pore-forming loop; both TRPV1 and TRPV4--and perhaps other TRPV family members--are influenced by glycosylation at this site. N-linked glycosylation, and the dynamic regulation of this process, substantially impacts function and targeting of TRP channels.

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.

Contribution of P2X 1 receptor intracellular basic residues to channel properties

The intracellular amino and carboxy termini of P2X receptors have been shown to contribute to the regulation of ATP evoked currents. In this study we produced, and expressed in Xenopus oocytes, individual alanine point mutants of positively charged amino acids (eight lysine, seven arginine and one histidine) in the intracellular domains of the human P2X 1 receptor. The majority of these mutations had no effect on the amplitude, time-course or rectification of ATP evoked currents. In contrast the mutant K367A was expressed at normal levels at the cell surface however ATP evoked currents were reduced by >99% and desensitised more rapidly demonstrating a role of K367 in channel regulation. This is similar to that previously described for T18A mutant channels. Co-expression of T18A and K367A mutant P2X 1 receptors produced larger ATP evoked responses than either mutant alone and suggests that these amino and carboxy terminal regions interact to regulate channel function.

Two PEST‐like motifs regulate Ca2+/calpain‐mediated cleavage of the CaVβ3 subunit and provide important determinants for neuronal Ca2+ channel activity

An increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation.

Who's Got the Calcium?

The human cardiac voltage-gated sodium channel (hH1) is essential for normal heart function, and mutations in the alpha subunit of the channel cause deadly arrhythmias. Some of these mutations are in a region that confers critical regulation of channel function by calcium, but exactly how calcium regulates the channel has been unclear. The hH1 protein contains an EF-hand calcium-binding motif that may bind calcium directly and an IQ-type calmodulin-binding motif that could confer calcium-dependent changes through changes in calmodulin. Shah et al. explored interactions between these domains to help clarify the roles of these direct and indirect responses to calcium in channel function. The authors characterized the binding properties of various channel domains and mutants by monitoring 15N-3H heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The EF-hand domain alone appeared to have a very low affinity for calcium (0.6 mM) that would be unlikely to have a physiological role. However, interaction of the IQ motif with the EF-hand domain enhanced calcium-binding affinity into the micromolar range. Contrary to expectations, a mutation in the EF-hand domain that causes arrhythmias in humans didn't alter calcium affinity but rather reduced affinity of the channel for binding calmodulin. On the other hand, a mutation in the IQ calmodulin-binding motif had little effect on affinity for calmodulin but reduced calcium-binding affinity. Also unusual was the finding that calcium-loaded calmodulin actually had lower affinity for an IQ motif-containing peptide than did calcium-free calmodulin. The authors propose that the effects of calcium can be explained as follows: In basal conditions, calmodulin is bound to the channel through the IQ motif. Increased concentrations of intracellular calcium weaken this interaction such that calmodulin is released, enabling the IQ motif to interact with the EF-hand domain, thus favoring calcium-dependent modulation of the channel through direct calcium binding. Further understanding of the intricate nature of these calcium-modulated domain interactions may reveal new therapeutic strategies to manage cardiac arrhythmias and other diseases associated with altered function of other members of this channel family in other cell types. V. N. Shah, T. L. Wingo, K. L. Weiss, C. K. Williams, J. R. Balser, W. J. Chazin, Calcium-dependent regulation of the voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors use a common molecular switch. Proc. Natl. Acad. Sci. U.S.A. 103 , 3592-3597 (2006). [Abstract] [Full Text]

The Cystic Fibrosis Transmembrane Conductance Regulator Is Regulated by a Direct Interaction with the Protein Phosphatase 2A

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.

Q/R Site Editing Controls Kainate Receptor Inhibition by Membrane Fatty Acids

RNA editing within the pore loop controls the pharmacology and permeation properties of ion channels formed by neuronal AMPA and kainate receptor subunits. Genomic sequences for the glutamate receptor 2 (GluR2) subunit of AMPA receptors and the GluR5 and GluR6 subunits of kainate receptors all encode a neutral glutamine (Q) residue within the channel pore that can be converted by RNA editing to a positively charged arginine (R). Receptors comprised of unedited subunits are permeable to calcium and display inwardly rectifying current-voltage relationships, because of blocking of outward current by intracellular polyamines. In contrast, receptors that include edited subunits conduct less calcium, resist polyamine block, and have relatively linear current-voltage relationships. We showed previously that cis-unsaturated fatty acids, including arachidonic acid and docosahexanoic acid, exert a potent block of native kainate receptors as well as homomeric recombinant receptors formed by transfection of heterologous cells with cDNA for the GluR6(R) subunit. Here, we show that fatty acid blockade of recombinant homomeric and heteromeric kainate receptors is strongly dependent on editing at the Q/R site. Recombinant channels that include unedited subunits exhibit significantly weaker block than channels made up of fully edited subunits. Inhibition of fully edited channels is equivalent at voltages from -70 to +40 mV and is noncompetitive, consistent with allosteric regulation of channel function.

Regulation of native GABAA receptors by PKC and protein phosphatase activity

Protein kinase C (PKC) modulation of ionotropic receptors is a common mechanism for regulation of channel function. The effects of PKC and phosphatase activation on native gamma-aminobutyric acid (GABA(A)) receptors in adult brain are unknown. Previous studies of recombinant GABA(A) receptors have provided evidence that PKC activation inhibits receptor function, whereas other studies suggest that PKC either increases or does not alter GABA(A) receptor function. The present study explored (a) the effects of PKC and phosphatase activity on GABA-mediated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes and (b) the effect of PKC activity on muscimol-induced loss of righting reflex (LORR) in adult rats. GABA(A) receptor function in vitro was measured by muscimol-induced (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The in vivo effect of PKC on GABA(A)-mediated function was measured by intracerebroventricular (i.c.v.) injection of 4-beta-phorbol-12,13-dibutyrate (PDBu) or calphostin C followed by determination of muscimol-induced LORR. Adenosine triphosphate (ATP) and PDBu produced a concentration-dependent and specific reduction in muscimol-stimulated (36)Cl(-) uptake that was blocked by the PKC inhibitor calphostin C. Both adenosine diphosphate and 4alphaPDBu were ineffective. Phosphatase inhibition produced similar inhibition of muscimol responses. Furthermore, i.c.v. administration of PDBu and calphostin C produced opposing effects on both the onset and the duration of muscimol-induced LORR in rats. The present study provides evidence that PKC activation reduces GABA(A) receptor function in native receptors both in vitro and in vivo. Phosphatase inhibitors decrease muscimol-mediated Cl(-) uptake in GABA(A) receptors demonstrating coordinated regulation of native receptors by PKC and phosphatases.

Coassembly of Two GluR6 Kainate Receptor Splice Variants within a Functional Protein Complex

Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

Spatial and Temporal Regulation of RACK1 Function and N-methyl-D-aspartate Receptor Activity through WD40 Motif-mediated Dimerization

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains.

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.

Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart.

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation-contraction coupling by providing a pathway for the release of Ca(2+) from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca(2+) and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIdelta. Quantitative assessment of RyR2 protein was performed by [(3)H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca(2+) release (Ca(2+) sparks) using confocal microscopy and (ii) Ca(2+)-sensitive [(3)H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 microM AIP Ca(2+) spark duration, frequency and width were decreased significantly. Similarly, 1 microM AIP decreased [(3)H]ryanodine binding. At 5 microM AIP, a more profound inhibition of Ca(2+) sparks and a decrease in [(3)H]ryanodine binding was observed. Separate measurements showed that AIP (1-5 microM) did not affect sarcoplasmic reticulum Ca(2+)-ATPase-mediated Ca(2+) uptake. These results suggest the existence of an endogenous CaMKIIdelta that associates directly with RyR2 and specifically modulates RyR2 activity.

The familial hemiplegic migraine mutation R192Q reduces G-protein-mediated inhibition of P/Q-type (Ca(V)2.1) calcium channels expressed in human embryonic kidney cells.

Familial hemiplegic migraine is associated with at least 13 different missense mutations in the alpha1A Ca(2+) channel subunit. Some of these mutations have been shown to affect the biophysical properties of alpha1A currents. To date, no study has examined the influence of such mutations on the G-protein regulation of channel function. Because G-proteins inhibit movement of the voltage sensor, we examined the effects of the R192Q mutation, which neutralizes a positive charge in the first S4 segment. Human wild-type (WT) or R192Q mutant channels were expressed in human embryonic kidney tsA-201 cells along with dopamine D2 receptors. Application of quinpirole induced fast (approximately 1 s), pertussis toxin-sensitive inhibition of alpha1A(WT) and alpha1A(R192Q) Ca(2+) currents, consistent with the activation of a membrane-delimited pathway. alpha1A(WT) Ca(2+) currents were inhibited by 62.9 +/- 0.9 % (n = 27), whereas alpha1A(R192Q) Ca(2+) currents were inhibited by only 47.9 +/- 1.8 % (n = 35; P < 0.001). Concentration-response analysis showed that only the extent of inhibition was affected, with no change in agonist potency (EC(50) = 1 nM). Prepulse facilitation, which is a characteristic of voltage-dependent inhibition, was also reduced by the R192Q mutation. However, the kinetics of facilitation and slow activation were not affected, suggesting that G-protein-Ca(2+) channel affinity was unchanged. These results show that the R192Q mutation reduces the G-protein inhibition of P/Q-type Ca(2+) channels, probably by altering mechanisms by which Gbetagamma subunit binding induces a change in channel gating. Altered G-protein modulation and the consequent reduced presynaptic inhibition may contribute to migraine attacks by favouring a persistent state of hyperexcitability.

P2X1 Receptor Subunit Contribution to Gating Revealed by a Dominant Negative PKC Mutant

The family of ATP-gated P2X receptor channels have a conserved protein kinase C site in the N-terminal intracellular domain. This site was disrupted in human P2X1 receptors by the mutation T18A. T18A mutants were expressed at normal levels in Xenopus oocytes; however, the peak current amplitude was reduced by >99% and showed ∼10 fold faster desensitisation in response to ATP than wild type (WT) receptors showed. P2X receptor subunits form functional trimeric channels. Co-expression of T18A and WT receptors (90:10 ratio) produced heteromeric T18A/WT channels with the rapid T18A time-course and an ∼90-fold increase in peak current amplitude compared to T18A. Similarly, T18A dominated the desensitisation phenotype of heteromeric channels composed of T18A and slowly desensitising K68A mutants. These results suggest that phosphorylation of P2X1 receptors has a dramatic effect on the time-course of the response and may provide a mechanism for regulating channel function.

Potassium channel structures.

The molecular basis of K+ channel function is universally conserved. K+ channels allow K+ flux and are essential for the generation of electric current across excitable membranes. K+ channels are also the targets of various intracellular control mechanisms, such that the suboptimal regulation of channel function might be related to pathological conditions. Because of the fundamental role of K+ channels in controlling membrane excitability, a structural understanding of their function and regulation will provide a useful framework for understanding neuronal physiology. Many recent physiological and crystallographic studies have led to new insights into the workings of K+ channels.

Ryanodine receptors, FKBP12, and heart failure

RyR2 function is regulated by highly conserved signaling pathways that modulate excitation-contraction (EC) coupling. cAMP dependent protein kinase (PKA) phosphorylation of RyR2 plays an important role in regulating channel function in response to stress signaled by the sympathetic nervous system (the classic "fight or flight response") (1). PKA phosphorylation of RyR2 induces dissociation of the regulatory protein FKBP12.6 resulting in channels with increased sensitivity to Ca2+-induced Ca2+ release. Under normal physiological conditions (no cardiac damage) PKA phosphorylation of RyR2 is part of an integrated physiological response that leads to increased EC coupling gain and increased cardiac output. PKA-hyperphosphorylation of RyR2 in failing hearts is a maladaptive response that results in depletion of FKBP12.6 from the RyR2 macromolecular complex and defective channel function (pathologically increased sensitivity to Ca2+-induced Ca2+ release) that may cause depletion of SR Ca2+ and diastolic release of SR Ca2+ that can initiate delayed after depolarizations (DADs) that trigger ventricular arrhythmias (1). RyR2 mutations in patients with catecholaminergic induced sudden cardiac death provide further evidence linking the sympathetic nervous system, RyR2 and ventricular arrhythmias (2-4). The chronic hyperadrenergic state of heart failure is associated with defective Ca2+ signaling in part due to PKA hyperphosphorylation of RyR2.

Ryanodine receptors, FKBP12, and heart failure.

RyR2 function is regulated by highly conserved signaling pathways that modulate excitation-contraction (EC) coupling. cAMP dependent protein kinase (PKA) phosphorylation of RyR2 plays an important role in regulating channel function in response to stress signaled by the sympathetic nervous system (the classic "fight or flight response") (1). PKA phosphorylation of RyR2 induces dissociation of the regulatory protein FKBP12.6 resulting in channels with increased sensitivity to Ca2+-induced Ca2+ release. Under normal physiological conditions (no cardiac damage) PKA phosphorylation of RyR2 is part of an integrated physiological response that leads to increased EC coupling gain and increased cardiac output. PKA-hyperphosphorylation of RyR2 in failing hearts is a maladaptive response that results in depletion of FKBP12.6 from the RyR2 macromolecular complex and defective channel function (pathologically increased sensitivity to Ca2+-induced Ca2+ release) that may cause depletion of SR Ca2+ and diastolic release of SR Ca2+ that can initiate delayed after depolarizations (DADs) that trigger ventricular arrhythmias (1). RyR2 mutations in patients with catecholaminergic induced sudden cardiac death provide further evidence linking the sympathetic nervous system, RyR2 and ventricular arrhythmias (2-4). The chronic hyperadrenergic state of heart failure is associated with defective Ca2+ signaling in part due to PKA hyperphosphorylation of RyR2.