Modulation Of Channel Activity Research Articles

Expression and functions of annexins in the kidney

This review article summarizes current knowledge about the locations and possible functions of annexin family members in the kidney. Beginning with an introduction on common structural and biochemical features as well as general functional characteristics of annexins, the paper focuses on individual members with documented and/or proposed physiological relevance for renal development, structure, and functions. Three main aspects of annexin function in kidney epithelia emerge from the available experimental data. First, annexins are required for membrane organization and membrane transport events required for the establishment/maintenance of epithelial polarity. Second, there is accumulating evidence of an association of annexins with ion channels, as membrane-guiding auxiliary proteins or modulators of channel activity. Last but not least, some annexins seem to work as extracellular autocrine modulators of receptor function under different physiological conditions.

Insulin Secretagogues, Sulfonylurea Receptors and KATP Channels

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.

The Antiarrhythmic Effect of n-3 Polyunsaturated Fatty Acids: Modulation of Cardiac Ion Channels as a Potential Mechanism

Sudden cardiac death remains one of the most serious medical challenges in Western countries. Increasing evidence in recent years has demonstrated that the n-3 polyunsaturated fatty acids (PUFAs) can prevent fatal ventricular arrhythmias in experimental animals and probably in humans. Dietary supplement of fish oils or intravenous infusion of the n-3 PUFAs prevents ventricular fibrillation caused by ischemia/reperfusion. Similar antiarrhythmic effects of these fatty acids are also observed in cultured mammalian cardiomyocytes. Based on clinical observations and experimental studies in vitro and in vivo, several mechanisms have been postulated for the antiarrhythmic effect of the n-3 PUFAs. The data from our laboratory and others have shown that the n-3 PUFAs are able to affect the activities of cardiac ion channels. The modulation of channel activities, especially voltage-gated Na(+) and L-type Ca(2+) channels, by the n-3 fatty acids may explain, at least partially, the antiarrhythmic action. It is not clear, however, whether one or more than one mechanism involves the beneficial effect of the n-3 PUFAs on the heart. This article summarizes our recent studies on the specific effects of the n-3 PUFAs on cardiac ion channels. In addition, the effect of the n-3 PUFAs on the human hyperpolarization-activated cyclic-nucleotide-modulated channel is presented.

A Calmodulin Binding Domain of RyR Increases Activation of Spontaneous Ca2+ Sparks in Frog Skeletal Muscle

The calmodulin C lobe binding region (residues 3614-3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614-3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614-3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614-3643 (0.2-3.0 microM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 microM and a maximal increase in frequency of approximately 5-fold. A peptide with a single amino acid substitution within R3614-3643 (L3624D) retained the ability to bind Ca(2+)-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614-3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.

AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner.

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.

Interaction of the Pacemaker Channel HCN1 with Filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

Folding of Active Calcium Channel β1b -Subunit by Size-exclusion Chromatography and Its Role on Channel Function

Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.

Molecular basis for modulation of recombinant alpha1beta2gamma2 GABAA receptors by protons.

We have previously shown that extracellular protons inhibit recombinant and native GABA(A) receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABA(A) receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant alpha1beta2gamma2 GABA(A) receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in alpha1(L264T)beta2gamma2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABA(A) receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of beta2 Y205 to F or alpha1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn(2+) sensitivity, suggesting that H(+) and Zn(2+) do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn(2+)-mediated inhibition of GABA(A) receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn(2+) modulation had no effect on pH modulation of GABA(A) receptors. Our results support the hypothesis that protons inhibit GABA(A) receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H(+) and Zn(2+) in GABA(A) receptors are distinct.

Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.

Cellular Actions of Gabapentin and Related Compounds on Cultured Sensory Neurones

In this review we outline the actions of gabapentin and pregabalin on the excitability of sensory dorsal root ganglion (DRG) neurones in culture and compare these effects with those seen in other neuronal and cultured cell preparations. We also consider the potential mechanisms of action of gabapentin and pregabalin that may contribute to their anti-nociceptive effects. Gabapentin and pregabalin have similar actions and at saturating concentrations do not have additive effects suggesting that they act at the same or closely associated sites. The only high affinity binding sites yet identified for these drugs are α2δ-subunits (types 1 and 2 but not 3 and 4) of voltage-activated Ca2+ channels. Consistent with this are the findings that gabapentin and pregabalin both attenuate Ca2+ influx through voltage-activated channels and these inhibitory actions may in part provide a mechanism by which multiple firing of action potentials in sensory neurones is reduced. However, it is clear that gabapentin and pregabalin have a number of novel characteristics. Although structural analogues of GABA they appear to act independently of GABA receptor activation in sensory neurones. The actions of gabapentin and pregabalin on sensory neurones are not consistent with selective effects on particular high voltage-activated Ca2+ channel subtypes (L, N, P, Q and R) yet a significant proportion (at least 50%) of the whole cell Ca2+ current is insensitive to these drugs. Cell culture conditions can alter Ca2+ channel subunit expression in such a way as to reduce sensitivity to gabapentin and this can be used to identify potential sites of action of this drug. Analysis of subunit mRNA in cell populations with different sensitivities to gabapentin surprisingly indicated that Ca2+ channel b2-subunits as well as α2δ-subunits may determine Ca2+ channel sensitivity to gabapentin in DRG neurones. In addition to Ca2+ channels, gabapentin and pregabalin modulate other whole cell currents, and in particular a delayed enhancement of voltage-dependent K+ conductances has been observed in cultured DRG neurones. The delayed and prolonged features of this enhancement of voltage-activated K+ currents may reflect gabapentin and pregabalin modulating channel activity via intracellular signalling pathways either via a surface receptor interaction leading to the production of a second messenger or by directly acting at intracellular sites. The wide variety of therapeutic effects achieved with gabapentin and pregabalin offer a considerable challenge when trying to determine cellular mechanisms of action of these apparently simple synthetic compounds. Keywords: gabapentin, sensory dorsal root, pregabalin, anti-nociceptive effects

Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function.

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.

A Novel TRPM2 Isoform Inhibits Calcium Influx and Susceptibility to Cell Death

TRPM2 is a Ca(2+)-permeable channel that is activated by oxidative stress and confers susceptibility to cell death. Here, an isoform of TRPM2 was identified in normal human bone marrow that consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Because of alternative splicing, a stop codon (TAG) is located at the splice junction between exons 16 and 17, resulting in deletion of the four C-terminal transmembrane domains, the putative calcium-permeable pore region, and the entire C terminus. This splice variant was found in other hematopoietic cells including human burst forming unit-erythroid-derived erythroblasts and TF-1 erythroleukemia cells. Endogenous expression of both the short form of TRPM2 (TRPM2-S) and the full length (TRPM2-L) was determined by reverse transcriptase-PCR, and localization of endogenous TRPM2 to the plasma membrane was demonstrated by confocal microscopy. Heterologous expression of TRPM2-S in HEK 293T cells demonstrated similar membrane localization as TRPM2-L, and coexpression of TRPM2-S did not alter the subcellular localization of TRPM2-L. The direct interaction of TRPM2-S with TRPM2-L was demonstrated with immunoprecipitation. H(2)O(2) induced calcium influx through TRPM2-L expressed in 293T cells. Coexpression of TRPM2-S suppressed H(2)O(2)-induced calcium influx through TRPM2-L. Furthermore, expression of TRPM2-S inhibited susceptibility to cell death and onset of apoptosis induced by H(2)O(2) in cells expressing TRPM2-L. These data demonstrate that TRPM2-S is an important physiologic isoform of TRPM2 and modulates channel activity and induction of cell death by oxidative stress through TRPM2-L.

New IP 3 Receptor Partner?

The inositol 1,4,5-trisphosphate receptor (IP 3 R) is a ligand-gated ion channel that mediates release of Ca 2+ from intracellular stores in response to stimuli that promote hydrolysis of phosphatidylinositol 4,5-bisphosphate. In this crucial role, the channel is, of course, highly regulated, and this occurs in part through interaction with other proteins. In a screen for new interaction partners of the IP 3 receptor, Ando et al. identified a protein they call IRBIT (for IP 3 R-binding protein released with inositol 1,4,5-trisphosphate), which has a number of intriguing properties. IRBIT looks like S -adenosylhomocysteine hydrolase, the enzyme that cleaves S -adenosylhomocysteine to form adenosine and homocysteine, but the authors could find no such enzymatic activity of IRBIT. IRBIT from extracts of rat cerebellar microsomes bound to the NH 2 -terminal portion of the IP 3 R in vitro, and the interaction was localized to the IP 3 -binding region of the IP 3 R. IRBIT contains numerous potential phosphorylation sites, and treatment of extracts with alkaline phosphatase prevented interaction of IRBIT with the IP 3 R. Immunoprecipitation of proteins from lysates of mouse cerebellum with an antibody to IRBIT provided evidence that the IP 3 R and IRBIT interact in vivo. The authors' original screen selected for proteins whose interaction with the IP 3 R was altered by binding of IP 3 , and further analysis of in vitro binding showed that concentrations of IP 3 estimated to be in the physiological range displaced IRBIT bound to the IP 3 R. The authors note that IRBIT displaced from the IP 3 R upon binding of IP 3 could generate another signal independent of the well-characterized Ca 2+ mobilization. Alternative roles for IRBIT in modulating channel activity, regulating susceptibility of the receptor to proteolytic degradation, or as a linker or scaffold are also possible. H. Ando, A. Mizutani, T. Matsu-ura, K. Mikoshiba, IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor. J. Biol. Chem. 278 , 10602-10612 (2003). [Abstract] [Full Text]

Long-term potentiation and long-term depression in hippocampal CA1 neurons of mice lacking the IP 3 type 1 receptor

To investigate the role in synaptic plasticity of Ca 2+ released from intracellular Ca 2+ stores, mice lacking the inositol 1,4,5-trisphosphate type 1 receptor were developed and the physiological properties, long-term potentiation, and long-term depression of their hippocampal CA1 neurons were examined. There were no significant differences in basic synaptic functions, such as membrane properties and the input/output relationship, between homozygote mutant and wild-type mice. Enhanced paired-pulse facilitation at interpulse intervals of less than 60 ms and enhanced post-tetanic potentiation were observed in the mutant mice, suggesting that the presynaptic mechanism was altered by the absence of the inositol 1,4,5-trisphosphate type 1 receptor. Long-term potentiation in the field-excitatory postsynaptic potentials induced by tetanus (100 Hz, 1 s) and the excitatory postsynaptic currents induced by paired stimulation in hippocampal CA1 pyramidal neurons under whole-cell clamp conditions were significantly greater in mutant mice than in wild-type mice. Homosynaptic long-term depression of CA1 synaptic responses induced by low-frequency stimulation (1 Hz, 500 pulses) was not significantly different, but heterosynaptic depression of the non-associated pathway induced by tetanus was blocked in the mutant mice. Both long-term potentiation and long-term depression in mutant mice were completely dependent on N-methyl- d-aspartate receptor activity. To rule out the possibility of an effect compensating for the lack of the inositol 1,4,5-trisphosphate type 1 receptor occurring during development, an anti-inositol 1,4,5-trisphosphate type 1 receptor monoclonal antibody that blocks receptor function was diffused into the wild-type cell through a patch pipette, and the effect of acute block of inositol 1,4,5-trisphosphate type 1 receptor on long-term potentiation was examined. Significant enhancement of long-term potentiation was observed compared with after control immunoglobulin G injection, suggesting that developmental redundancy was not responsible for the increase in long-term potentiation amplitude observed in the mutant mouse. The properties of channels that could be involved in long-term potentiation induction were examined using whole-cell recording. N-methyl- d-aspartate currents were significantly larger in mutant mice than in wild-type mice only between holding potentials of −60 and −80 mV. We conclude that inositol 1,4,5-trisphosphate type 1 receptor activity is not essential for the induction of synaptic plasticity in hippocampal CA1 neurons, but appears to negatively regulate long-term potentiation induction by mild modulation of channel activities.

Nitric Oxide, NOC-12, and S-Nitrosoglutathione Modulate the Skeletal Muscle Calcium Release Channel/Ryanodine Receptor by Different Mechanisms: AN ALLOSTERIC FUNCTION FOR O2 INS-NITROSYLATION OF THE CHANNEL

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) contains approximately 50 thiols per subunit. These thiols have been grouped according to their reactivity/responsiveness toward NO, O(2), and glutathione, but the molecular mechanism enabling redox active molecules to modulate channel activity is poorly understood. In the case of NO, very low concentrations (submicromolar) activate RyR1 by S-nitrosylation of a single cysteine residue (Cys-3635), which resides within a calmodulin binding domain. S-Nitrosylation of Cys-3635 only takes place at physiological tissue O(2) tension (pO(2); i.e. approximately 10 mm Hg) but not at pO(2) approximately 150 mm Hg. Two explanations have been offered for the loss of RyR1 responsiveness to NO at ambient pO(2), i.e. Cys-3635 is oxidized by O(2) versus O(2) subserves an allosteric function (Eu, J. P., Sun, J. H., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509). Here we report that the NO donors NOC-12 and S-nitrosoglutathione both activate RyR1 by release of NO but do so independently of pO(2). Moreover, NOC-12 activates the channel by S-nitrosylation of Cys-3635 and thereby reverses channel inhibition by calmodulin. In contrast, S-nitrosoglutathione activates RyR1 by oxidation and S-nitrosylation of thiols other than Cys-3635 (and calmodulin is not involved). Our results suggest that the effect of pO(2) on RyR1 S-nitrosylation is exerted through an allosteric mechanism.

Sulfhydryl Modulation of K + Channels in Rat Ventricular Myocytes

G. J. Rozanski and Z. Xu. Sulfhydryl Modulation of K + Channels in Rat Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2002) 34, 1623–1632. Oxidative stress markedly alters protein function through redox modification of sulfhydryl groups present in cysteine residues. To explore the role of redox state in modulating cardiac K + channels, this study examined the effects of sulfhydryl modifiers on the repolarizing transient outward current (I to) in voltage-clamped myocytes from rat ventricle. Oxidized glutathione (GSSG; 5 mM), an endogenous disulfide that specifically reacts with protein sulfhydryls, decreased maximum I to amplitude from baseline by 49% when added to the external solution ( P<0.05) and by 27% during internal dialysis ( P<0.05). The membrane-impermeable disulfide, 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) did not alter I to when added to the external solution, but it decreased current amplitude by 31% during internal dialysis ( P<0.05). GSSG-mediated I to inhibition varied in a frequency- and voltage-dependent manner, consistent with a state-dependent blocking mechanism. This phenomenon was also observed in myocytes internally dialyzed with DTNB or Cd 2+, which also covalently binds to free sulfhydryls. Inhibition of I to by GSSG was not reversed by washout alone, consistent with the stable nature of covalently-modified sulfhydryl groups. However, when myocytes pretreated with GSSG were dialyzed with the reducing agent dithiothreitol, I to amplitude increased significantly by 42% ( P<0.05). These data suggest that α-subunits underlying I to, or associated proteins, have one or more sulfhydryl groups within the cytoplasmic domain that directly modulate channel activity in response to changes in cell redox state. Redox modulation of I to channels may be an important post-translational mechanism contributing to acute changes in cardiac repolarization under conditions of oxidative stress, such as ischemia and reperfusion.

Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells.

Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In "normal" ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.

Cloning and Expression of the Human T-Type Channel Cav3.3: Insights into Prepulse Facilitation

The full-length human Cav3.3 (α1I) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3′ untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5mM Ca2+ as charge carrier. From a holding potential of −100mV, step depolarizations elicited inward currents with an apparent threshold of −70mV, a peak of −30mV, and reversed at +40mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Cav3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Cav3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Cav3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100mV, 200ms) increased the fraction of channels that gated at low voltages. In contrast, human Cav3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Cav3.3 is similar to high voltage-activated Ca2+ channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.

Modulation ofDrosophilaSlowpoke Calcium-Dependent Potassium Channel Activity by Bound Protein Kinase A Catalytic Subunit

Drosophila Slowpoke (dSlo) calcium-dependent potassium channels bind directly to the catalytic subunit of cAMP-dependent protein kinase (PKAc). We demonstrate here that coexpression of PKAc with dSlo in mammalian cells results in a dramatic decrease of dSlo channel activity. This modulation requires catalytically active PKAc but is not mediated by phosphorylation of S942, the only PKA consensus site in the dSlo C-terminal domain. dSlo binds to free PKAc but not to the PKA holoenzyme that includes regulatory subunits and is inactive. Activators of endogenous PKA that stimulate dSlo phosphorylation, but do not produce detectable PKAc binding to dSlo, do not modulate channel function. Furthermore, the catalytically inactive PKAc mutant does bind to dSlo but does not modulate channel activity. These results are consistent with the hypothesis that both binding of active PKAc to dSlo and phosphorylation of dSlo or some other protein are necessary for channel modulation.

Dual Action of PIP 2

Activation of some TRP-type calcium ion channels requires activation of phospholipase C (PLC) and hydrolysis of its substrate phosphatidylinositol 4,5-bisphosphate (PIP 2 ). This cascade is initiated when certain heterotrimeric guanine nucleotide-biding protein (G protein)-coupled receptors are activated. But Runnels et al. report the deviant behavior of one family member, TRPM7. Activation of either a G-protein-coupled receptor or a receptor tyrosine kinase actually inhibited TRPM7 through the same signaling pathway of PIP 2 hydrolysis through PLC. TRPM7 coimmunoprecipitated with PLC-β 2 when the two proteins were overexpressed in cultured cells. The inhibitory effect of PIP 2 on TRPM7 channel currents was blocked by the presence of antibodies against PIP 2 . Activation of the G-protein-coupled receptor for lysophophatidic acid in cardiac myocytes also inhibited endogenous TRPM7 activity. ATP and magnesium have also been implicated in regulating TRPM7 but it is presently unclear how any of these molecules directly modulate channel activity. The authors propose that the activating or inhibitory nature of PIP 2 on TRP-type channels may depend on the proximity of specific PLC isoforms to TRP channels. L. W. Runnels, L. Xie, D. E. Clapham, The TRPM7 channel is inactivated by PIP 2 hydrolysis. Nature Cell Biol . 4 , 32-336 (2002). [Online Journal]