Tubulin as a Binding Partner of the Heag2 Voltage-Gated Potassium Channel

The aim of this work was to investigate interactions of the human ether-a-go-go channel heag2 with human brain proteins. For this, we used heag2-GST fusion proteins in pull-down assays with brain proteins and mass spectrometry, as well as coimmunoprecipitation. We identified tubulin and heat shock 70 proteins as binding to intracellular C-terminal regions of the channel. To study functional effects, heag2 channels were expressed in Xenopus laevis oocytes for two-electrode voltage clamping. Coexpression of alpha-tubulin or the application of colchicine significantly prolonged channel activation times. Application at different times of colchicine gave similar results. The data suggest that colchicine application and tubulin expression do not affect heag2 trafficking and that tubulin may associate with the channel to cause functional effects. Coexpression of heat shock 70 proteins had no functional effect on the channel. The role of tubulin in the cell cytoskeleton suggests a link for the heag2 channel in tubulin-dependent physiological functions, such as cellular proliferation.     

Molecular regions responsible for differences in activation between heag channels

The ether-a-go-go potassium channels heag1 and heag2 are highly homologous; however, the activation properties between the two channels are different. We have studied the molecular regions that determine differences in activation properties by making chimeras between the two channels, expressing them in oocytes, and recording currents with two-electrode voltage-clamp. The activation time course has an initial sigmoidal component dependent on the Cole-Moore shift, followed by a faster component. We show that not only is the extreme N terminus involved in differences between heag1 and heag2 channels, but also the PAS domain itself. Also multiple regions of the membrane-spanning part of the channel appear to be involved, with different regions involved for the early and late time courses, reflecting their different mechanisms. The later time course involved S1 and P-S6 regions. Taken together, our data show that activation involves multiple regions of the N terminal region and membrane-spanning regions of the channel.     

Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

Intracellular regions of potassium channels: Kv2.1 and heag

Intracellular regions of voltage-gated potassium channels often comprise the largest part of the channel protein, and yet the functional role of these regions is not fully understood. For the Kv2.1 channel, although there are differences in activation kinetics between rat and human channels, there are, for instance, no differences in movement of the S4 region between the two channels, and indeed our mutagenesis studies have identified interacting residues in both the N- and C -terminal intracellular regions that are responsible for these functional effects. Furthermore, using FRET with fluorescent-tagged Kv2.1 channels, we have shown movement of the C-termini relative to the N-termini during activation. Such interactions and movements of the intracellular regions of the channel appear to form part of the channel gating machinery. Heag1 and heag2 channels also display differing activation properties, despite their considerable homology. By a chimeric approach, we have shown that these differences in activation kinetics are determined by multiple interacting regions in the N-terminus and membrane-spanning regions. Furthermore, alanine mutations of many residues in the C-terminal cyclic nucleotide binding domain affect activation kinetics. The data again suggest interacting regions between N- and C- termini that participate in the conformational changes during channel activation. Using a mass-spectrometry approach, we have identified α-tubulin and a heat shock protein as binding to the C-terminus of the heag2 channel, and α-tubulin itself has functional effects on channel activation kinetics. Clearly, the intracellular regions of these ion channels (and most likely many other ion channels too) are important regions in determining channel function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

A novel spliced variant of the epithelial Na+ channel delta-subunit in the human brain

The amiloride-sensitive epithelial Na+ channel regulates Na+ homeostasis in cells. Recently, we described that the delta-subunit is a candidate molecule for a pH sensor in the human brain. Here, an N-terminal spliced variant of the delta-subunit is cloned from human brain, and designated as the delta2-subunit, which is expressed with the original delta-subunit (delta1-subunit) at the same level in the human brain. Functional analyses revealed that the physiological and pharmacological properties (interaction with accessory betagamma-subunits, activation by acidic pH, amiloride sensitivity) of the delta2-subunit were similar to those of the delta1-subunit. In conclusion, the activities of both subunits may be involved in the mechanism underlying pH sensing in the human brain.     

Differential effects of beta 1 and beta 2 subunits on BK channel activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.     

Functional and biochemical evidence for G-protein-gated inwardly rectifying K+ (GIRK) channels composed of GIRK2 and GIRK3

G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.     

Eicosanoids inhibit the G-protein-gated inwardly rectifying potassium channel (Kir3) at the Na+/PIP2 gating site

We previously showed that activation of the human endothelin A receptor (HETAR) by endothelin-1 (Et-1) selectively inhibits the response to mu opioid receptor (MOR) activation of the G-protein-gated inwardly rectifying potassium channel (Kir3). The Et-1 effect resulted from PLA2 production of an eicosanoid that inhibited Kir3. In this study, we show that Kir3 inhibition by eicosanoids is channel subunit-specific, and we identify the site within the channel required for arachidonic acid sensitivity. Activation of the G-protein-coupled MOR by the selective opioid agonist D-Ala(2)Glyol, enkephalin, released Gbetagamma that activated Kir3. The response to MOR activation was significantly inhibited by Et-1 activation of HETAR in homomeric channels composed of either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were substantially less sensitive. Domain deletion and channel chimera studies suggested that the sites within the channel required for Et-1-induced inhibition were within the region responsible for channel gating. Mutation of a single amino acid in the homomeric Kir3.1 to produce Kir3.1(F137S)(N217D) dramatically increased the channel sensitivity to arachidonic acid and Et-1 treatment. Complementary mutation of the equivalent amino acid in Kir3.4 to produce Kir3.4(S143T)(D223N) significantly reduced the sensitivity of the channel to arachidonic acid- and Et-1-induced inhibition. The critical aspartate residue required for eicosanoid sensitivity is the same residue required for Na(+) regulation of PIP(2) gating. The results suggest a model of Kir3 gating that incorporates a series of regulatory steps, including Gbetagamma, PIP(2), Na(+), and arachidonic acid binding to the channel gating domain.     

Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4

We present the cloning and characterization of two novel calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4, that are enriched in the testis and brain, respectively. We compare and contrast the steady state and kinetic properties of these beta subunits with the previously cloned mouse beta1 (mKCNMB1) and the human beta2 subunit (hKCNMB2). Once inactivation is removed, we find that hKCNMB2 has properties similar to mKCNMB1. hKCNMB2 slows Hslo1 channel gating and shifts the current-voltage relationship to more negative potentials. hKCNMB3 and hKCNMB4 have distinct effects on slo currents not observed with mKCNMB1 and hKCNMB2. Although we found that hKCNMB3 does interact with Hslo channels, its effects on Hslo1 channel properties were slight, increasing Hslo1 activation rates. In contrast, hKCNMB4 slows Hslo1 gating kinetics, and modulates the apparent calcium sensitivity of Hslo1. We found that the different effects of the beta subunits on some Hslo1 channel properties are calcium-dependent. mKCNMB1 and hKCNMB2 slow activation at 1 microM but not at 10 microM free calcium concentrations. hKCNMB4 decreases Hslo1 channel openings at low calcium concentrations but increases channel openings at high calcium concentrations. These results suggest that beta subunits in diverse tissue types fine-tune slo channel properties to the needs of a particular cell.     

Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.     

A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.     

Mutation of divergent region 1 alters caffeine and Ca(2+) sensitivity of the skeletal muscle Ca(2+) release channel (ryanodine receptor)

Replacement of amino acids 4187-4628 in the skeletal muscle Ca(2+) release channel (skeletal ryanodine receptor (RyR1)), including nearly all of divergent region 1 (amino acids 4254-4631), with the corresponding cardiac ryanodine receptor (RyR2) sequence leads to increased sensitivity of channel activation by caffeine and Ca(2+) and to decreased sensitivity of channel inactivation by elevated Ca(2+) (Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126). In further investigations, this region was subdivided by the construction of new chimeras, and alterations in channel function were detected by measurement of the caffeine dependence of in vivo Ca(2+) release and the Ca(2+) dependence of [(3)H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC(50) value for activation by caffeine, and RF10c (4557-4628) had a higher EC(50) value, whereas the EC(50) value for chimera RF10b (4382-4556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca(2+), whereas RF10a was less sensitive to inactivation by Ca(2+), implicating RF10b and RF10c in Ca(2+) activation and RF10a in Ca(2+) inactivation. Deletion of much of divergent region 1 sequence to create mutant Delta4274-4535 led to higher caffeine and Ca(2+) sensitivity of channel activation and to lower Ca(2+) sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca(2+), and ryanodine binding sites are not located in amino acids 4274-4535. Nevertheless, the properties of the deletion and chimeric mutants demonstrate that amino acids 4274-4535 and three shorter sequences in this region (F10a, amino acids 4187-4381; F10b, 4382-4556; and F10c, 4557-4628) in RyR1 modulate Ca(2+) and caffeine sensitivity of the Ca(2+) release channel.     

Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K+ Channel,GIRK, in Xenopus Oocytes

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K⁺ channel, GIRK, is activated by the βγ subunits of G_i proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by G_i/G_o-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the G_q class, inhibited the channel''s activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca²⁺ chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-μ. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a G_i/G_o protein. The observed modulations may be involved in the mGluRs'' effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to G_q are inefficient in activating GIRK.     

Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as both a chloride channel and an epithelial transport regulator, interacting with Na(+) (epithelial sodium channel), Cl(-), renal outer medullary potassium channel(+), and H(2)O channels and some exchangers (i.e. Na(+)/H(+)) and co-transporters (Na(+)-HCO(3)(minus sign), Na(+)-K(+)-2Cl(-)). Acid-sensitive ion channels (ASICs), members of the epithelial sodium channel/degenerin superfamily, were originally cloned from neuronal tissue, and recently localized in epithelia. Because CFTR has been immunocytochemically and functionally identified in rat, murine, and human brain, the regulation of ASICs by CFTR was tested in oocytes. Our observations show that the proton-gated Na(+) current formed by the heteromultimeric ASIC1a/2a channel was up-regulated by wild type but not by Delta F508-CFTR. In contrast, the acid-gated Na(+) current associated with either the homomultimeric ASIC1a or ASIC2a channel was not influenced by wild type CFTR. The apparent equilibrium dissociation constant for extracellular Na(+) for ASIC1a/2a was increased by CFTR, but CFTR had no effect on the gating behavior or acid sensitivity of ASIC1a/2a. CFTR had no effect on the pH activation of ASIC1a/2a. We conclude that wild type CFTR elevates the acid-gated Na(+) current of ASIC1a/2a in part by altering the kinetics of extracellular Na(+) interaction.     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library

A full-length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1-encoded protein, when expressed in Xenopus oocytes, gated a 4-aminopyridine (4-AP)-sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage-dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.     

Identification of a putative voltage-gated Ca2+ channel as a key regulator of elicitor-induced hypersensitive cell death and mitogen-activated protein kinase activation in rice

Elicitor-triggered transient membrane potential changes and Ca2+ influx through the plasma membrane are thought to be important during defense signaling in plants. However, the molecular bases for the Ca2+ influx and its regulation remain largely unknown. Here we tested effects of overexpression as well as retrotransposon (Tos17)-insertional mutagenesis of the rice two-pore channel 1 (OsTPC1), a putative voltage-gated Ca(2+)-permeable channel, on a proteinaceous fungal elicitor-induced defense responses in rice cells. The overexpressor showed enhanced sensitivity to the elicitor to induce oxidative burst, activation of a mitogen-activated protein kinase (MAPK), OsMPK2, as well as hypersensitive cell death. On the contrary, a series of defense responses including the cell death and activation of the MAPK were severely suppressed in the insertional mutant, which was complemented by overexpression of the wild-type gene. These results suggest that the putative Ca(2+)-permeable channel determines sensitivity to the elicitor and plays a role as a key regulator of elicitor-induced defense responses, activation of MAPK cascade and hypersensitive cell death.     

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).     

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) alpha subunits alone. Because native BKCa channel complexes may include the auxiliary subunit beta1, we investigated whether beta1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with beta1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75 mV) as compared with that with alpha alone (-30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of beta1. Oxidation of cysteine and methionine residues within beta1 was not involved in these potentiated effects because expression of mutant beta1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type beta1. Unlike the results with alpha alone, oxidation by Ch-T caused a significant acceleration of channel activation only when beta1 was present. The beta1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming alpha subunit are greatly amplified by the presence of beta1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within beta1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with beta1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.