James L. Kenyon

Cyclic GMP-dependent Protein Kinase Activates Cloned BKCa Channels Expressed in Mammalian Cells by Direct Phosphorylation at Serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the α-subunit of the large conductance Ca2+-activated K+ channel,cslo-α, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-α exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) ofcslo-α channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Iα to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-α in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-α by direct phosphorylation at serine 1072.

Unitary Cl− Channels Activated by Cytoplasmic Ca2+ in Canine Ventricular Myocytes

Recent whole-cell studies have shown that Ca(2+)-activated Cl- currents contribute to the Ca(2+)-dependent 4-aminopyridine-insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl(-)-sensitive currents are activated by Ca2+ release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl- to identify 4-aminopyridine-resistant unitary Ca(2+)-activated Cl- channels, Ca2+ applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl- selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca2+ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca2+ (ranging from pCa > or = 6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NPo, where N is the number of channels opened and Po is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NPo on cytoplasmic Ca2+ yielded an apparent Kd of 150.2 mumol/L. At pCa 3, an average channel density of approximately equal to 3 microns-2 was estimated. Calculations based on these estimates of cytoplasmic Ca2+ sensitivity and channel current amplitude and density suggest that these small-conductance Cl- channels contribute significant whole-cell membrane current in response to changes in intracellular Ca2+ within the physiological range. We suggest that these small-conductance Ca(2+)-activated Cl- channels underlie the transient Ca(2+)-activated 4-aminopyridine-insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.

Contribution of delayed rectifier potassium currents to the electrical activity of murine colonic smooth muscle

1 We used intracellular microelectrodes to record the membrane potential (Vm) of intact murine colonic smooth muscle. Electrical activity consisted of spike complexes separated by quiescent periods (Vm≈−60 mV). The spike complexes consisted of about a dozen action potentials of approximately 30 mV amplitude. Tetraethylammonium (TEA, 1–10 mM) had little effect on the quiescent periods but increased the amplitude of the action potential spikes. 4‐Aminopyridine (4‐AP, ? 5 mM) caused continuous spiking. 3 Voltage clamp of isolated myocytes identified delayed rectifier K+ currents that activated rapidly (time to half‐maximum current, 11.5 ms at 0 mV) and inactivated in two phases (τf= 96 ms, τs= 1.5 s at 0 mV). The half‐activation voltage of the permeability was −27 mV, with significant activation at −50 mV. 4 TEA (10 mM) reduced the outward current at potentials positive to 0 mV. 4‐AP (5 mM) reduced the early current but increased outward current at later times (100–500 ms) consistent with block of resting channels relieved by depolarization. 4‐AP inhibited outward current at potentials negative to −20 mV, potentials where TEA had no effect. 6 Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvβ1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. 7 We find that TEA‐sensitive delayed rectifier currents are important determinants of action potential amplitude but not rhythmicity. Delayed rectifier currents sensitive to 4‐AP are important determinants of rhythmicity but not action potential amplitude.

Expression of cystic fibrosis transmembrane regulator Cl- channels in heart.

Cyclic AMP (cAMP)-dependent chloride channels modulate changes in resting membrane potential and action potential duration in response to autonomic stimulation in heart. A growing body of evidence suggests that there are marked similarities in the properties of the cAMP-dependent chloride channels in heart and cystic fibrosis transmembrane regulator (CFTR) chloride channels found in airway epithelia or in cells expressing the CFTR gene product. We isolated poly A+ mRNA from rabbit ventricle and converted it to cDNA for amplification using the polymerase chain reaction (PCR). A fragment corresponding to the nucleotide-binding domain 1 (NBD1) of the CFTR transcript was cloned. Comparison of the amino acid sequence of NBD1 of human CFTR with the deduced sequence of the rabbit heart PCR product indicated 98% identity. Northern blot analysis, using the heart amplification product as a cDNA probe, demonstrated expression of homologous transcripts in human atrium, guinea pig and rabbit ventricle, and dog pancreas. Xenopus oocytes injected with poly A+ mRNA extracted from rabbit and guinea pig ventricle or dog pancreas expressed robust time-independent chloride currents in response to an elevation of cAMP. We conclude that CFTR chloride channels are expressed in heart and are responsible for the observed cAMP-dependent chloride conductance.

Functional and molecular expression of a voltage-dependent K+ channel (Kv1.1) in interstitial cells of Cajal

Located within the gastrointestinal (GI) musculature are networks of cells known as interstitial cells of Cajal (ICC). ICC are associated with several functions including pacemaker activity that generates electrical slow waves and neurotransmission regulating GI motility. In this study we identified a voltage‐dependent K+ channel (Kv1.1) expressed in ICC and neurons but not in smooth muscle cells. Transcriptional analyses demonstrated that Kv1.1 was expressed in whole tissue but not in isolated smooth muscle cells. Immunohistochemical co‐localization of Kv1.1 with c‐kit (a specific marker for ICC) and vimentin (a specific marker of neurons and ICC) indicated that Kv1.1‐like immunoreactivity (Kv1.1‐LI) was present in ICC and neurons of GI tissues of the dog, guinea‐pig and mouse. Kv1.1‐LI was not observed in smooth muscle cells of the circular and longitudinal muscle layers. Kv1.1 was cloned from a canine colonic cDNA library and expressed in Xenopus oocytes. Pharmacological investigation of the electrophysiological properties of Kv1.1 demonstrated that the mamba snake toxin dendrotoxin‐K (DTX‐K) blocked the Kv1.1 outward current when expressed as a homotetrameric complex (EC50= 0.34 nm). Other Kv channels were insensitive to DTX‐K. When Kv1.1 was expressed as a heterotetrameric complex with Kv1.5, block by DTX‐K dominated, indicating that one or more subunits of Kv1.1 rendered the heterotetrameric channel sensitive to DTX‐K. In patch‐clamp experiments on cultured murine fundus ICC, DTX‐K blocked a component of the delayed rectifier outward current. The remaining, DTX‐insensitive current (i.e. current in the presence of 10−8m DTX‐K) was outwardly rectifying, rapidly activating, non‐inactivating during 500 ms step depolarizations, and could be blocked by both tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP). In conclusion, Kv1.1 is expressed by ICC of several species. DTX‐K is a specific blocker of Kv1.1 and heterotetrameric channels containing Kv1.1. This information is useful as a means of identifying ICC and in studies of the role of delayed rectifier K+ currents in ICC functions.

Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.

Molecular identification of a component of delayed rectifier current in gastrointestinal smooth muscles

Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.2 is expressed in smooth muscle cells found in all regions of the canine gastrointestinal (GI) tract and in several vascular tissues. Injection of Kv2.2 mRNA into Xenopus oocytes resulted in the expression of a slowly activating K+ current (time to half maximum current, 97 ± 8.6 ms) mediated by 15 pS (symmetrical K+) single channels. The current was inhibited by tetraethylammonium (IC50 = 2.6 mM), 4-aminopyridine (IC50 = 1.5 mM at +20 mV), and quinine (IC50 = 13.7 μM) and was insensitive to charybdotoxin. Low concentrations of quinine (1 μM) were used to preferentially block the slow component of the delayed rectifier current in native colonic myocytes. These data suggest that Kv2.2 may contribute to this current in native GI smooth muscle cells.Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.2 is expressed in smooth muscle cells found in all regions of the canine gastrointestinal (GI) tract and in several vascular tissues. Injection of Kv2.2 mRNA into Xenopus oocytes resulted in the expression of a slowly activating K+ current (time to half maximum current, 97 +/- 8.6 ms) mediated by 15 pS (symmetrical K+) single channels. The current was inhibited by tetraethylammonium (IC50 = 2.6 mM), 4-aminopyridine (IC50 = 1.5 mM at +20 mV), and quinine (IC50 = 13.7 microM) and was insensitive to charybdotoxin. Low concentrations of quinine (1 microM) were used to preferentially block the slow component of the delayed rectifier current in native colonic myocytes. These data suggest that Kv2.2 may contribute to this current in native GI smooth muscle cells.

Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II

1 The kinetics of inactivation of delayed rectifier K+ current in murine colonic myocytes differed in amphotericin‐permeabilized patch and conventional patch clamp. The difference was accounted for by Ca2+ buffering. 2 Calcium‐calmodulin‐dependent protein kinase II (CaMKII) inhibitors increased the rate of inactivation and slowed recovery from inactivation of the outward current. This was seen in single steps and in the envelope of the current tails. The effect was largely on the TEA‐insensitive component of current. 3 Dialysis of myocytes with autothiophosphorylated CaMKII slowed inactivation. This effect was reversed by addition of CaMKII inhibitor. 4 Antibodies revealed CaMKII‐like immunoreactivity in murine colonic myocytes and other cells. Immunoblots identified a small protein with CaMKII‐like immunoreactivity in homogenates of colonic muscle. 5 We conclude that CaMKII regulates delayed rectifier K+ currents in murine colonic myocytes. The changes in the delayed rectifier current may participate in the Ca2+‐dependent regulation of gastrointestinal motility.

Novel voltage-dependent non-selective cation conductance in murine colonic myocytes

Two components of voltage‐gated, inward currents were observed from murine colonic myocytes. One component had properties of L‐type Ca2+ currents and was inhibited by nicardipine (5 × 10−7m). A second component did not ‘run down’ during dialysis and was resistant to nicardipine (up to 10−6m). The nicardipine‐insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. This low‐voltage‐activated current (ILVA) was resolved with step depolarizations positive to ‐60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half‐inactivation was ‐65 mV. Inactivation and activation time constants at ‐45 mV were 86 and 15 ms, respectively. The half‐recovery time from inactivation was 98 ms at ‐45 mV. ILVA peaked at ‐40 mV and the current reversed at 0 mV. I lva was inhibited by Ni2+ (IC50= 1.4 × 10−5m), mibefradil (10−6 to 10−5m), and extracellular Ba2+. Replacement of extracellular Na+ with N‐methyl‐d‐glucamine inhibited ILVA and shifted the reversal potential to ‐7 mV. Increasing extracellular Ca2+ (5 × 10−3m) increased the amplitude of ILVA and shifted the reversal potential to +22 mV. ILVA was also blocked by extracellular Cs+ (10−4m) and Gd3+ (10−6m). Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. We conclude that the second component of voltage‐dependent inward current in murine colonic myocytes is not a ‘T‐type’ Ca2+ current but rather a novel, voltage‐gated non‐selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.