Shift In Voltage Dependence Of Activation Research Articles

Association with the Auxiliary Subunit PEX5R/Trip8b Controls Responsiveness of HCN Channels to cAMP and Adrenergic Stimulation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key modulators of neuronal activity by providing the depolarizing cation current I(h) involved in rhythmogenesis, dendritic integration, and synaptic transmission. These tasks critically depend on the availability of HCN channels, which is dynamically regulated by intracellular cAMP; the range of this regulation, however, largely differs among neurons in the mammalian brain. Using affinity purification and high-resolution mass spectrometry, we identify the PEX5R/Trip8b protein as the beta subunit of HCN channels in the mammalian brain. Coassembly of PEX5R/Trip8b affects HCN channel gating in a subtype-dependent and mode-specific way: activation of HCN2 and HCN4 by cAMP is largely impaired, while gating by phosphoinositides and basal voltage-dependence remain unaffected. De novo expression of PEX5R/Trip8b in cardiomyocytes abolishes beta-adrenergic stimulation of HCN channels. These results demonstrate that PEX5R/Trip8b is an intrinsic auxiliary subunit of brain HCN channels and establish HCN-PEX5R/Trip8b coassembly as a mechanism to control the channels' responsiveness to cyclic nucleotide signaling.

Charge-dependent And Isoform-specific Interactions Between ProTxii And T-type Calcium Channels

ProTxII, peptide toxin isolated from the venom of the tarantula Thrixopelma puriens, modifies voltage-dependent activation of both T-type calcium (Ca) channels and voltage-gated sodium (Na) channels. In the presence of ProTxII (5μM) the voltage at half maximal activation (V1/2) of the CaV3.1 isoform is shifted positive (>25mV) and maximum conductance (Gmax) decreases (∼50%). Interestingly, the toxin's effects on this channel were completely precluded in the presence of high extracellular divalent concentrations indicating a role for surface charge-like, electrostatic interactions with the channel. Several mutant toxins in which individual basic residues were neutralized were tested for activity on CaV3.1. Three of these mutants, R13Q, R22A, and K28A, significantly disrupted the ability of the toxin to both shift channel activation and decrease Gmax. Two other mutations: K4Q and K14A, showed minimal or no effect, thus indicating an important yet specific role of charge in ProTxII's interaction with CaV3.1. The gating kinetics of T-type Ca channels varies among the three known isoforms suggesting there might be differences in the gating structures and, therefore, potential gating modifier toxin interaction surfaces as well. In CaV3.3, 5μM ProTxII reduced Gmax by approximately 60%, similar to what was seen for CaV3.1. However, unlike in CaV3.1, this concentration of toxin produced only a minimal shift in voltage dependent activation (2mV). These results suggest significant differences in the extracellular surface of T-type Ca channels across isoforms, particularly in terms of surface charge distribution close to one or more of the channels’ voltage sensors.

Glutamate Transporters Regulate Extrasynaptic NMDA Receptor Modulation of Kv2.1 Potassium Channels

Delayed-rectifier Kv2.1 potassium channels regulate somatodendritic excitability during periods of repetitive, high-frequency activity. Recent evidence suggests that Kv2.1 channel modulation is linked to glutamatergic neurotransmission. Because NMDA-type glutamate receptors are critical regulators of synaptic plasticity, we investigated NMDA receptor modulation of Kv2.1 channels in rodent hippocampus and cortex. Bath application of NMDA potently unclustered and dephosphorylated Kv2.1 and produced a hyperpolarizing shift in voltage-dependent activation of voltage-sensitive potassium currents (I(K)). In contrast, driving synaptic activity in Mg2+-free media to hyperactivate synaptic NMDA receptors had no effect on Kv2.1 channels, and moderate pentylenetetrazole-induced seizure activity in adult mice did not dephosphorylate hippocampal Kv2.1 channels. Selective activation of extrasynaptic NMDA receptors unclustered and dephosphorylated Kv2.1 channels and produced a hyperpolarizing shift in neuronal I(K). In addition, inhibition of glutamate uptake rapidly activated NMDA receptors and dephosphorylated Kv2.1 channels. These observations demonstrate that regulation of intrinsic neuronal activity by Kv2.1 is coupled to extrasynaptic but not synaptic NMDA receptors. These data support a novel mechanism for glutamate transporters in regulation of neuronal excitability and plasticity through extrasynaptic NMDA receptor modulation of Kv2.1 channels.

ISA Channel Complexes Include Four Subunits Each of DPP6 and Kv4.2

Kv4 potassium channels produce rapidly inactivating currents that regulate excitability of muscles and nerves. To reconstitute the neuronal A-type current I(SA), Kv4 subunits assemble with DPP6, a single transmembrane domain accessory subunit. DPP6 alters function-accelerating activation, inactivation, and recovery from inactivation-and increases surface expression. We sought here to determine the stoichiometry of Kv4 and DPP6 in complexes using functional and biochemical methods. First, wild type channels formed from subunit monomers were compared with channels carrying subunits linked in tandem to enforce 4:4 and 4:2 assemblies (Kv4.2-DPP6 and Kv4.2-Kv4.2-DPP6). Next, channels were overexpressed and purified so that the molar ratio of subunits in complexes could be assessed by direct amino acid analysis. Both biophysical and biochemical methods indicate that I(SA) channels carry four subunits each of Kv4.2 and DPP6.

Mutation I136V Alters Electrophysiological Properties of the NaV1.7 Channel in a Family with Onset of Erythromelalgia in the Second Decade

BackgroundPrimary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na+ channel NaV1.7 have been linked with primary erythromelalgia. Recently, a new substitution NaV1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9–22 years, with onset at 17 years of age or later in 5 of 7 family members), with relatively slow progression (8–10 years) to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1) of domain I (DI) in contrast to all NaV1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III.ResultsIn this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents.ConclusionThe I136V substitution in NaV1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of NaV1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.

NADPH binding to β-subunit regulates inactivation of voltage-gated K + channels

Ancillary β-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvβ proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvβ1.3 binds NADPH ( K d = 1.8 ± 0.02 μM) and NADP + ( K d = 5.5 ± 0.9 μM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K d NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvβ1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvβ-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvα–β interaction. Inactivation of Kv currents by Kvβ:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvα and β subunits and for Kvβ-induced inactivation of Kv currents.

Modulatory effect of auxiliary β1 subunit on Nav1.3 voltage-gated sodium channel expressed in Xenopus oocyte

homologous and form single transmembrane glycoproteins with short intracellular loops and immunoglobulin-like extracellular segments. The association of the various α subtypes with different combinations of auxiliary β subunits creates the possibility of additional molecular and functional complexity for neuronal sodium channels. Nav1.1, Nav1.2, Nav1.3 and Nav1.6 are predominantly expressed in the central nervous system while Nav1.7, Nav1.8 and Nav1.9 are principally found in the peripheral nervous system. 2 The Nav1.3 α subtype has attracted interest recently because its expression is dramatically up-regulated in nociceptive sensory neurons after peripheral nerve damage, perhaps contributing to the pathophysiology of neuropathic pain. 3 Theoretically, no matter how the expression of the Nav1.3 α subunit is changed in neuropathic pain, the function of the Nav1.3 α subtype should be modulated by β subunits. The aim of this study was to examine the effects of cloned rat β1 subunit on Nav1.3 function.

C-terminal Domain of Kv4.2 and Associated KChIP2 Interactions Regulate Functional Expression and Gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.

H‐89 inhibits transient outward and inward rectifier potassium currents in isolated rat ventricular myocytes

1. Voltage clamp was used to investigate the effects of N-[2-p-bromo-cinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a potent inhibitor of PKA, on transient outward K(+) current (I(to)) and inward rectifying K(+) current (I(K1)) in rat cardiac muscle. 2. Initial experiments, performed using descending voltage ramps, showed that H-89 inhibited both the outward and inward ramp currents in a concentration-dependent manner at concentrations between 5 and 60 micromol l(-1). A similar degree of inhibition was observed when I(to) and I(K1) were recorded using square wave depolarising and hyperpolarising voltage steps, respectively. 3. The IC(50) was 35.8 micromol l(-1) for I(to) and 27.8 micromol l(-1) for I(K1) compared to 5.4 micromol l(-1) for L-type Ca(2+) current (I(Ca)). The Hill coefficients for I(to), I(K1) and I(Ca) were -1.97, -1.60 and -1.21, respectively. In addition to inhibiting I(to) amplitude, H-89 also accelerated the time to peak and the rate of voltage-dependent inactivation so that the time course of I(to) was abbreviated. 4. Paired-pulse protocols were performed to study the effects of H-89 on steady-state activation and inactivation as well as recovery from voltage-dependent inactivation. H-89 produced a concentration-dependent rightward shift in voltage-dependent activation but had no significant effect on steady-state inactivation. Recovery from voltage-dependent inactivation was delayed, although this was only visible at the highest concentration (60 micromol l(-1)) used. In experiments investigating the effects of elevated cyclic AMP, the beta-adrenergic agonist isoprenaline and the phosphatase inhibitor calyculin A had no major effects on I(to) or I(K1). 6. Data suggest that the effects of H-89 on K(+) currents are more complex than simple inhibition of PKA-mediated phosphorylation.

Functional Roles of Ca v 1.3(α 1D ) Calcium Channels in Atria

Background— Previous data suggest that L-type Ca 2+ channels containing the Ca v 1.3(α 1D ) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca v 1.2(α 1C ) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Ca v 1.3 Ca 2+ channel–deficient mice ( Ca v 1.3 −/− ) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Ca v 1.3(α 1D ) Ca 2+ channel in the atria of Ca v 1.3 −/− mice. Methods and Results— We obtained new evidence to demonstrate that there is significant expression of Ca v 1.3 Ca 2+ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca 2+ currents ( I Ca,L ) recorded from single, isolated atrial myocytes from Ca v 1.3 −/− mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the I Ca,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Ca v 1.3 compared with Ca v 1.2 Ca 2+ channel subunits in a heterologous expression system. The lack of Ca v 1.3 Ca 2+ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I Ca,L in atrial myocytes. In addition, the Ca v 1.3 -null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Ca v 1.3 versus Ca v 1.2 by in situ hybridization and immunofluorescence confocal microscopy. Conclusions— Using gene-targeted deletion of the Ca v 1.3 Ca 2+ channel, we established the differential distribution of Ca v 1.3 Ca 2+ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Ca v 1.3 Ca 2+ channel in atrial tissues.

Retigabine: Bending Potassium Channels to Our Will

The New Anticonvulsant Retigabine Favors Voltage-dependent Opening of the Kv7.2 (KCNQ2) Channel by Binding to Its Activation Gate Wuttke TV, Seebohm G, Bail S, Maljevic S, Lerche H Mol Pharmacol 2005;67:1009–1017 Retigabine (RTG) is an anticonvulsant drug with a novel mechanism of action. It activates neuronal KCNQ-type K+ channels by inducing a large hyperpolarizing shift of steady-state activation. To identify the structural determinants of KCNQ channel activation by RTG, we constructed a set of chimeras by using the neuronal KV7.2 ( KCNQ2) channel, which is activated by RTG, and the cardiac KV7.1 ( KCNQ1) channel, which is not affected by this drug. Substitution of either the S5 or the S6 segment in KV7.2 by the respective parts of KV7.1 led to a complete loss of activation by RTG. Trp236 in the cytoplasmic part of S5 and the conserved Gly301 in S6 (KV7.2), considered as the gating hinge (Ala336 in KV7.1), were found to be crucial for the RTG effect: mutation of these residues could either knock out the effect in KV7.2 or restore it partially in KV7.1/KV7.2 chimeras. We propose that RTG binds to a hydrophobic pocket formed upon channel opening between the cytoplasmic parts of S5 and S6 involving Trp236 and the channel's gate, which could well explain the strong shift in voltage-dependent activation.

The New Anticonvulsant Retigabine Favors Voltage-Dependent Opening of the Kv7.2 (KCNQ2) Channel by Binding to Its Activation Gate

Retigabine (RTG) is an anticonvulsant drug with a novel mechanism of action. It activates neuronal KCNQ-type K(+) channels by inducing a large hyperpolarizing shift of steady-state activation. To identify the structural determinants of KCNQ channel activation by RTG, we constructed a set of chimeras using the neuronal K(v)7.2 (KCNQ2) channel, which is activated by RTG, and the cardiac K(v)7.1 (KCNQ1) channel, which is not affected by this drug. Substitution of either the S5 or the S6 segment in K(v)7.2 by the respective parts of K(v)7.1 led to a complete loss of activation by RTG. Trp236 in the cytoplasmic part of S5 and the conserved Gly301 in S6 (K(v)7.2), considered as the gating hinge (Ala336 in K(v)7.1), were found to be crucial for the RTG effect: mutation of these residues could either knockout the effect in K(v)7.2 or restore it partially in K(v)7.1/K(v)7.2 chimeras. We propose that RTG binds to a hydrophobic pocket formed upon channel opening between the cytoplasmic parts of S5 and S6 involving Trp236 and the channel's gate, which could well explain the strong shift in voltage-dependent activation.

Unique modulation of L-type Ca2+channels by short auxiliary β1dsubunit present in cardiac muscle

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Ca(v)beta subunits. The Ca(v)beta(1d) isoform encodes a putative protein composed of the amino-terminal half of the full-length Ca(v)beta(1) isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel alpha1-subunit, the alpha-binding pocket. The present study investigated whether the Ca(v)beta(1d) subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Ca(v)beta subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Ca(v)beta(1d) identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Ca(v)beta(1b) subunit promoted membrane trafficking of the pore-forming alpha1C (Ca(v)1.2)-subunit to the surface membrane, whereas the Ca(v)beta(1d) subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca(v)beta(1d) with the alpha1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca(v)beta(1b) coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca(v)beta(1b) or Ca(v)beta(1d) significantly increased mean open probability four- to fivefold relative to the alpha1C-channels alone, but only Ca(v)beta(1b) coexpression increased the number of channels observed per patch. In conclusion, the Ca(v)beta(1d) isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.

Smooth Muscle-selective Alternatively Spliced Exon Generates Functional Variation in Cav1.2 Calcium Channels

Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel alpha(1)-subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Ca(v)1.2 alpha(1)-subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9(*) in Ca(v)1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9(*) localized the intense expression of 9(*)-containing Ca(v)1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9(*)-containing Ca(v)1.2 channels in HEK293 cells demonstrated -9 and -11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the +/-exon 9(*) splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming alpha(1)-subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca(v)1.2 alpha(1)-subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.

Cyclic AMP regulates the HERG K+ channel by dual pathways

Lethal cardiac arrhythmias are a hallmark of the hereditary Long QT syndrome (LQTS), a disease produced by mutations of cardiac ion channels [1]. Often these arrhythmias are stress-induced, suggesting a relationship between beta-adrenergic activation of adenylate cyclase and cAMP-dependent alteration of one or more of the ion channels involved in LQTS. Second messengers modulate ion channel activity either by direct interaction or through intermediary kinases and phosphatases. Here we show that the second messenger cAMP regulates the K(+) channel mutated in the LQT2 form of LQTS, HERG [2], both directly and indirectly. Activation of cAMP-dependent protein kinase (PKA) causes phosphorylation of HERG accompanied by a rapid reduction in current amplitude, acceleration of voltage-dependent deactivation, and depolarizing shift in voltage-dependent activation. In a parallel pathway, cAMP directly binds to the HERG protein with the opposing effect of a hyperpolarizing shift in voltage-dependent activation. The summation of cAMP-mediated effects is a net diminution of the effective current, but when HERG is complexed with with the K(+) channel accessory proteins MiRP1 or minK, the stimulatory effects of cAMP are favored. These findings provide a direct link between stress and arrhythmia by a unique mechanism where a single second messenger exerts complex regulation of an ion channel via two distinct pathways.

Role of a novel KCa opener in regulating K+ channels of hypoxic human pulmonary vascular cells.

Hypoxic pulmonary vasoconstriction (HPVC) is mediated, in part, via membrane depolarization and inhibition of K+ channels. We recently observed that the naturally occurring steroid dehydroepiandrosterone (DHEA) reversed and prevented HPVC in isolated perfused and ventilated ferret lungs. In the current study, we investigated the effects of DHEA on the major K+ channels of chronically hypoxic human pulmonary smooth-muscle cells (HPSMC). K+ channels were recorded by using the patch-clamp technique in whole-cell and single-channel configurations. Single-channel recordings were performed in inside-out and outside-out excised patches, and in intact HPSMC in cell-attached configuration. Using whole-cell current recording, chronic hypoxia decreased the high-amplitude, high-noise, and charybdotoxin-sensitive Ca2+-dependent K+ channels (KCa). DHEA reversed the effect of chronic hypoxia on KCa, but had no effect on the low-amplitude, low-noise, and 4-aminopyridine-sensitive delayed rectifying K+ channels. In the cell-attached configuration, chronic hypoxia caused a decrease in KCa sensitivity to membrane potential (Em). DHEA reversed the effect of hypoxia on KCa sensitivity to Em and caused a mean of 40-mV left shift in voltage-dependent activation of KCa. DHEA increased KCa activation from both sides of membrane patches of hypoxic HPSMC via a cyclic adenosine monophosphate- and cyclic guanosine monophosphate-independent pathway. We concluded that DHEA is a novel KCa opener of the human pulmonary vasculature.

Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells.

We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.

Regulation of Ca(2+)-activated K+ channels by protein kinase A and phosphatase inhibitors.

Many proteins including ion channels are regulated by phosphorylation. We tested the effect of 10 U/ml catalytic subunit protein kinase A on 260-pS Ca(2+)-activated K+ channels in excised inside-out membrane patches from freshly dispersed smooth muscle cells of the canine proximal colon. At +50 mV with 10(-7) M Ca2+ and -50 mV with 10(-6) M Ca2+, open probability of the channels was increased to 270 +/- 48% of control (n = 12). This increase was due to a shift in voltage-dependent activation by 13.9 +/- 3.2 mV (n = 3) to more negative potentials. Protein kinase A in the absence of ATP had no effect on channel activity (n = 3). Regulation by phosphorylation must be accompanied by dephosphorylation. We tested the effect of two potent inhibitors of protein phosphatases, calyculin A and okadaic acid. Application of 10(-9) to 10(-6) M of each inhibitor in the presence of protein kinase A further increased open probability by up to 250%. Calyculin A appeared to be less effective in increasing open probability than okadaic acid, suggesting that the phosphatase involved is neither type 1, 2A, nor 2B. Calyculin A in the absence of protein kinase A was ineffective. These data suggest that endogenous phosphatases are found in excised membrane patches and that a balance between phosphorylation and dephosphorylation may provide an important control of colonic motility.

Muscarinic suppression of Ca2+-dependent K current in colonic smooth muscle.

Acetylcholine (ACh) increases the amplitude and duration of colonic electrical slow waves. This suggests that ACh either increases an inward current or suppresses an outward current. The latter hypothesis was tested in whole cell voltage-clamp experiments performed on freshly dispersed smooth muscle cells from canine proximal colon. Addition of ACh (10(-5) M) to solutions bathing cells reduced time-dependent outward currents elicited by depolarizing test pulses in the range of -45 to +30 mV. Analysis of tail currents showed that ACh caused a 10- to 15-mV positive shift in voltage-dependent activation. When cells were pretreated with 10(-6) M nifedipine to abolish the Ca2+-dependent component of the outward current, the reduction of outward current by ACh was blocked. Single-channel experiments were performed to determine whether ACh had a direct effect on Ca2+-activated K channels. ACh, 10(-5) M, added to bath and pipette solutions caused a positive shift in voltage-dependent activation in on-cell experiments. This effect of ACh on Ca2+-activated K channels provides a mechanism for the effects of muscarinic, excitatory stimulation of circular muscle of the colon.