Biomedical subjects

J L Kenyon

Publications and source records attributed to J L Kenyon.

At least 19 recordsLinked to original sources

PubMed2006

Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5' truncated cDNA.

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.

Amino Acid Sequence↗

PubMed2003

An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell.

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca2+ in a spherical cell in the presence of a fixed Ca2+ buffer and two diffusible Ca2+ buffers, one of which is considered to be a fluorescent Ca2+ indicator. We modeled Ca2+ diffusion during and after Ca2+ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca2+ activity ([Ca2+]avg) rises to a high peak and starts to decline promptly on the termination of Ca2+ influx. We compared [Ca2+]avg with predictions of ratiometric Ca2+ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca2+ ([Ca2+]avg,M1). Method 2 sums the measured number of moles of Ca2+ in each of the N compartments and divides by the volume of the cell ([Ca2+]avg,M2). [Ca2+]avg,M1 peaks well after the termination of Ca2+ influx at a value substantially less than [Ca2+]avg because the summed signals do not reflect the averaged free Ca2+ if the signals come from compartments containing gradients in free Ca2+ spanning nonlinear regions of the relationship between free Ca2+ and the fluorescence signals. In contrast, [Ca2+]avg,M2 follows [Ca2+]avg closely.

Adrenal Glands↗

PubMed2002

Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton.

The regulation of cardiac delayed rectifier potassium (Kv) currents by cAMP-dependent protein kinase (PKA) contributes to the control of blood pressure and heart rate. We investigated the modulation by PKA and protein phosphatases of cloned Kv1.5 channels expressed in Xenopus laevis oocytes. Exposure of oocytes to activators of PKA (100 nM forskolin, 1 mM 8-bromo-cAMP, or 1 mM 3-isobutyl-1-methylxanthine) had no effect on the amplitude of Kv1.5 currents. Inhibition of PKA by injection of protein kinase A inhibitor peptide or exposure to myristoylated protein kinase A inhibitor peptide (M-PKI; 100 nM) reduced currents mediated by Kv1.5. M-PKI also reduced the amplitude of currents mediated by mutated Kv1.5 channels in which the COOH terminal PKA phosphorylation sites and PSD-95, Disc-large, and ZO-1-binding domain were removed. The reduction of Kv1.5 currents by M-PKI was attenuated by inhibition of actin polymerization by 1 microM cytochalasins B and D, but was not affected by 10 microM phalloidin (stabilizes actin filaments) or 50 microM colchicine (disrupts microtubules). Treatment of oocytes with antisense oligonucleotides against alpha-actinin-2 abolished the reduction in Kv1.5 current by M-PKI. These observations suggest that Kv1.5 currents are activated by endogenous PKA in "resting" oocytes and that inhibition of PKA activity reveals the action of endogenous phosphatases. Indeed, injection of alkaline phosphatase reduced currents mediated by Kv1.5. Further preincubation of oocytes with 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor) abolished the reduction in Kv1.5 currents by M-PKI. We conclude that currents encoded by Kv1.5 are regulated by PKA and protein tyrosine phosphatase and that this regulation requires an intact actin cytoskeleton and alpha-actinin-2.

Actinin↗

PubMed2001

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

1. 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. 2. 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. 3. 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 (EC(50) = 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. 4. 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(-8) M 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). 5. 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.

Animals↗

PubMed2001

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

1. Two components of voltage-gated, inward currents were observed from murine colonic myocytes. One component had properties of L-type Ca(2+) currents and was inhibited by nicardipine (5 x 10(-7) M). A second component did not 'run down' during dialysis and was resistant to nicardipine (up to 10(-6) M). The nicardipine-insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. 2. This low-voltage-activated current (I(LVA)) 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. I(LVA) peaked at -40 mV and the current reversed at 0 mV. 3. I(LVA) was inhibited by Ni(2+) (IC(50) = 1.4 x 10(-5) M), mibefradil (10(-6) to 10(-5) M), and extracellular Ba(2+). Replacement of extracellular Na(+) with N-methyl-D-glucamine inhibited I(LVA) and shifted the reversal potential to -7 mV. Increasing extracellular Ca(2+) (5 x 10(-3) M) increased the amplitude of I(LVA) and shifted the reversal potential to +22 mV. I(LVA) was also blocked by extracellular Cs(+) (10(-4) M) and Gd(3+) (10(-6) M). 4. Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. 5. We conclude that the second component of voltage-dependent inward current in murine colonic myocytes is not a 'T-type' Ca(2+) 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.

Action Potentials↗

PubMed2001

Alosetron and the rapid component of delayed rectifying potassium current in cardiac cells.

Some drugs acting on 5-hydroxytryptamine receptors inhibit the rapid component of delayed rectifying potassium currents (I(Kr)) in cardiac muscle cells. This is associated with lengthening of the QT interval in the cardiac cycle and can lead to fatal arrhythmias. We investigated whether alosetron, a novel 5HT3 antagonist proposed for treatment of irritable bowel syndrome (IBS), blocks I(Kr) in guinea pig cardiac myocytes. I(Kr) was isolated under whole-cell voltage clamp, and was identified by its sensitivity to the selective I(Kr) antagonist E4031. Cisapride (10(-6) M) inhibited the E4031-sensitive current while alosetron (10(-10)-10(-6) M) had no effect on I(Kr). We also found that alosetron did not inhibit I(Ks). Therefore, use of alosetron for treatment of IBS should not be confounded by long QT syndrome.

Animals↗

PubMed2001

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

8,11,14-Eicosatrienoic Acid↗

PubMed2001

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C.

Localized Ca(2+) transients resulting from inositol trisphosphate (IP(3))-dependent Ca(2+) release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca(2+) transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca(2+) ([Ca(2+)](o)) reduced localized Ca(2+) transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca(2+) transients and STOCs showed increased coupling strength between Ca(2+) transients and STOCs when [Ca(2+)](o) was reduced. Gd(3+) (10 microM) did not affect Ca(2+) transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca(2+) influx, 1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca(2+) transients. Phorbol 12-myristate 13-acetate (1 microM) reduced STOC amplitude and frequency but did not affect Ca(2+) transients. 4alpha-Phorbol (1 microM) had no effect on STOCs or Ca(2+) transients. Single channel studies indicated that large-conductance Ca(2+)-activated K(+) (BK) channels were inhibited by a Ca(2+)-dependent PKC. In summary 1) Ca(2+) release from IP(3) receptor-operated stores activates Ca(2+)-activated K(+) channels; 2) Ca(2+) influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca(2+) sensitivity of BK channels, reducing the coupling strength between localized Ca(2+) transients and BK channels.

Animals↗

PubMed2001

Phorbol ester-induced inhibition of potassium currents in rat sensory neurons requires voltage-dependent entry of calcium.

The whole cell patch-clamp technique was used to examine the effects of protein kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents (I(K)) in cultured capsaicin-sensitive neurons isolated from dorsal root ganglia from embryonic rat pups and grown in culture. PDBu, in a concentration- and time-dependent manner, reduced I(K) measured at +60 mV by approximately 30% if the holding potential (V(h)) was -20 or -47 mV but had no effect if V(h) was -80 mV. The PDBu-induced inhibition of I(K) was blocked by pretreatment with the PKC inhibitor bisindolylmaleimide I and I(K) was unaffected by 4-alpha phorbol, indicating that the suppression of I(K) was mediated by PKC. The inhibition of I(K) by 100 nM PDBu at a V(h) of -50 mV was reversed over several minutes if V(h) was changed to -80 mV. In addition, intracellular perfusion with 5 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or pretreatment with omega-conotoxin GVIA or Cd(2+)-Ringer, but not nifedipine, prevented the PDBu-induced suppression of I(K) at -50 mV, suggesting that a voltage-dependent influx of calcium through N-type calcium channels was necessary for the activation of PKC. The potassium channel blockers tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 3 mM and 30 microM) reduced I(K), but only TEA attenuated the ability of PDBu to further inhibit the current, suggesting that the I(K) modified by PDBu was sensitive to TEA. Interestingly, in the presence of 3 mM or 30 microM 4-AP, 100 nM PDBu inhibited I(K) when V(h) was -80 mV. Thus 4-AP promotes the capacity of PDBu to reduce I(K) at -80 mV. We find that activation of PKC inhibits I(K) in rat sensory neurons and that voltage-dependent calcium entry is necessary for the development and maintenance of this inhibition.

4-Aminopyridine↗

PubMed2000

The reversal potential of Ca(2+)-activated Cl(-) currents indicates that chick sensory neurons accumulate intracellular Cl(-).

In order to establish the physiological role of the Ca(2+)-activated Cl(-) current (I(Cl(Ca))) of chick primary afferent neurons, I measured the reversal potential of this current using either the amphotericin perforated patch technique (that alters intracellular Cl(-)) or the gramicidin perforated patch technique (that does not perturb intracellular Cl(-)). In the amphotericin experiments at 35 degrees C, I(Cl(Ca)) reversed at the Cl(-) equilibrium potential (E(Cl)=-24 mV) set by the superfusate (147 mM Cl(-)) and the pipette solution (60 mM Cl(-)). In contrast, in the gramicidin experiments at 35 degrees C, I(Cl(Ca)) reversed at -42+/-2 mV, midway between E(Cl) of the solutions and E(Cl) expected if Cl(-) were passively distributed. Thus the gramicidin perforated patch technique monitors Cl(-) currents without perturbing intracellular Cl(-). Further, the data imply that chick dorsal root ganglia (DRG) neurons actively accumulate Cl(-). I(Cl(Ca)) reversed at the same potential (-46+/-3 mV) at 20 degrees C indicating that the non-equilibrium distribution of Cl(-) is maintained at the lower temperature. Thus, I(Cl(Ca)) is a depolarizing current that can contribute to the after-depolarization in chick DRG neurons and thereby alter Ca(2+) influx.

Amphotericin B↗

PubMed2000

Amplitude histograms can identify positively but not negatively coupled channels.

We investigated the ability of amplitude distributions to determine if the gating of a pair of channels is coupled. These distributions are expressed as probability density amplitude histograms with peaks corresponding to zero, one, or two open channels. If the channels gate independently, the areas under these peaks (A, B, and C, respectively) determine the open probabilities of the two channels (p(1) and p(2)). Manivannan et al. (Biophys J 1994;61:216) showed that if Delta=B(2)/AC was less than 4 then the channel gating is coupled. We defined a similar parameter, D=(B(2)/4)-AC. If D<0 then channel gating is coupled. However, amplitude histograms with D0 are consistent with both independent and coupled gating. We further present a simple model in which channels are assumed to be identical and can be positively or negatively coupled. Here, amplitude histograms determine q=(B+2C)/2 (open probability of the coupled channels) and r=-D (the coupling parameter). Thus, positively coupled channels (r0) produce amplitude histograms with D<0 whereas negatively coupled channels (r<0) produce amplitude histograms with D0.

Animals↗

PubMed2000

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons.

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.

Amphotericin B↗

PubMed2000

Kir3.1/3.2 encodes an I(KACh)-like current in gastrointestinal myocytes.

Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(KACh) could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to beta-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 microM) reversibly activated a Ba(2+)-sensitive and inwardly rectifying K(+) current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.

Acetylcholine↗

PubMed2000

Ryanodine-sensitive stores regulate the excitability of AH neurons in the myenteric plexus of guinea-pig ileum.

Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHP(slow)) in the soma. The role of intracellular Ca(2+) ([Ca(2+)](i)) and ryanodine-sensitive Ca(2+) stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca(2+)](i) was approximately 200 nM. Depolarizing current pulses that elicited APs evoked AHP(slow) and an increase in [Ca(2+)](i), with similar time courses. The amplitudes and durations of AHP(slow) and the Ca(2+) transient were proportional to the number of evoked APs, with each AP increasing [Ca(2+)](i) by approximately 50 nM. Ryanodine (10 microM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca(2+) transient and AHP(slow) over the range of APs tested (1-15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca(2+)](i) of approximately 30 nM Ca(2+) via CICR. This indicates that CICR amplifies Ca(2+) influx. Similar changes in [Ca(2+)](i) and AHP(slow) were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca(2+)](i) and not the source of the Ca(2+) is the determinant of the magnitude of AHP(slow). Furthermore, lowering of free [Ca(2+)](i), either by reducing extracellular Ca(2+) or injecting high concentrations of Ca(2+) buffer, induced depolarization, increased excitability, and abolition of AHP(slow). In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca(2+)](i) and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.

Action Potentials↗

PubMed1999

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

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 alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha 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) of cslo-alpha 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-Ialpha 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-alpha 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-alpha by direct phosphorylation at serine 1072.

Base Sequence↗

PubMed1999

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 approximately -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. 2. 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 (tauf = 96 ms, taus = 1.5 s at 0 mV). The half-activation voltage of the permeability was -27 mV, with significant activation at -50 mV. 3. 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. 4. Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvbeta1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. 5. 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.

4-Aminopyridine↗

PubMed1999

Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit kv2.2 in brain stem and hypothalamic neurons.

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.

Angiotensin II↗