Kir Currents Research Articles

Human neoplastic mesothelial cells express voltage-gated sodium channels involved in cell motility

Given the pivotal role of ion channels in neoplastic transformation, the aim of the present study has been to assess possible differences in the expression patterns of voltage-gated monovalent cationic (Na+ and K+) currents between normal and neoplastic mesothelial cells (NM, MPM, respectively), and to evaluate the role of specific ion channels in mesothelioma cells proliferation, apoptosis, and motility. To achieve this aim, membrane currents expressed in NM and MPM cells derived from surgically-removed human specimens were investigated by means of patch-clamp electrophysiology. NM cells were found to express three main classes of K+ currents, which were defined as KIR, maxiKCa, and KV currents on the basis of their biophysical and pharmacological properties. Each of these K+ currents was absent in MPM cells; by contrast, MPM cells revealed the novel appearance of tetrodotoxin (TTX)-sensitive voltage-gated Na+ currents undetected in normal mesothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR analysis of MPM cells transcripts showed significant expression of the mRNAs encoding for NaV1.2, and NaV1.6, and NaV1.7 (and less so for NaV1.3, NaV1.4, and NaV1.5) main voltage-gated sodium channel (VGSC) α-subunit(s). Interestingly, blockade of VGSCs with TTX decreased mesothelioma cell migration in in vitro motility assays; on the other hand, TTX failed to interfere with cell viability, proliferation, and apoptosis progression triggered by UV exposure. In summary, the results of the present study suggest that VGSCs expression in MPM cells may favor the increased motility of the neoplastic cells, a phenotypic feature often associated with the malignant phenotype.

The protein kinase A inhibitor, H-89, directly inhibits K ATP and Kir channels in rabbit coronary arterial smooth muscle cells

The effects of the protein kinase A (PKA) inhibitor H-89 on ATP-sensitive K + (K ATP) and inward rectifier K + (Kir) currents were examined in rabbit coronary arterial smooth muscle cells using the patch clamp technique. The H-89, in a dose-dependent manner, inhibited K ATP and Kir currents with apparent K d values of 1.19 ± 0.18 and 3.78 ± 0.37 μM, respectively. H-85, which is considered as an inactive form of H-89, inhibited K ATP and Kir currents, similar to the result of H-89. K ATP and Kir currents were not affected by either Rp-8-CPT-cAMPs, which is a membrane-permeable selective PKA inhibitor, or KT 5720, which is also known as a PKA inhibitor. Also, these two drugs did not significantly alter the effects of H-89 on the K ATP and Kir currents. These results suggest that H-89 directly inhibits the K ATP and Kir currents of rabbit coronary arterial smooth muscle cells independently of PKA inhibition.

Functional expression of Kir4.1 channels in spinal cord astrocytes

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild-type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.

Basic Science

Abstract Laura A. Jansen, Erik J. Uhlmann, Peter B. Crino, David H. Gutmann, and Michael Wong Individuals with Tuberous Sclerosis Complex (TSC) frequently suffer from intractable epilepsy. To gain insight into the causes of epilepsy in TSC, we previously developed a mouse model of TSC with inactivation of the Tsc1 gene selectively in brain astrocytes (Tsc1GFAPCKO mice). Astrocytes, the main category of glial cells in brain, are important supporting cells in the brain, and also have direct effects on brain physiology, development, and repair. These mice develop progressive seizures, suggesting that astrocyte dysfunction may be involved in the development of epilepsy in TSC. Since one of the important functions of astrocytes is to limit sudden elevations of extracellular potassium in the brain, which would lead to neuronal excitability, in this study we investigated the hypothesis that impairment of potassium uptake may contribute to the development of seizures in Tsc1GFAPCKO mice. Astrocytes take up potassium from the extracellular space via a membrane channel, called the Kir channel. Cultured astrocytes from Tsc1GFAPCKO mice exhibited reduced Kir potassium currents and decreased expression of specific Kir channel protein subunits. mRNA expression of the same Kir subunits was also reduced in astrocytes from Tsc1GFAPCKO mice. Furthermore, we showed that the impairment in Kir channel function was reversed with drugs (roscovitine and retinoic acid) that modulate cell signaling pathways implicated in TSC. Lastly, hippocampal slices from Tsc1GFAPCKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced seizure-like activity. In conclusion, impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to increased neuronal excitability and the development of epilepsy in a mouse model of TSC.

Epileptogenesis and Reduced Inward Rectifier Potassium Current in Tuberous Sclerosis Complex‐1–Deficient Astrocytes

Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.

Endothelin-1 Inhibits Inward Rectifier K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells Through Protein Kinase C

We studied inward rectifier K+ (Kir) channels in smooth muscle cells isolated from rabbit coronary arteries. In cells from small- (<100 microm, SCASMC) and medium-diameter (100 approximately 200 microm, MCASMC) coronary arteries, Kir currents were clearly identified (11.2 +/- 0.6 and 4.2 +/- 0.6 pA pF at -140 mV in SCASMC and MCASMC, respectively) that were inhibited by Ba(2+) (50 microm). By contrast, a very low Kir current density (1.6 +/- 0.4 pA pF) was detected in cells from large-diameter coronary arteries (>200 microm, LCASMC). The presence of Kir2.1 protein was confirmed in SCASMC in a Western blot assay. Endothelin-1 (ET-1) inhibited Kir currents in a dose-dependent manner. The inhibition of Kir currents by ET-1 was abolished by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (100 nM) or GF 109203X (1 microm). The PKC activators phorbol 12,13-dibutyrate (PDBu) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced Kir currents. The ETA-receptor inhibitor BQ-123 prevented the ET-1-induced inhibition of Kir currents. The amplitudes of the ATP-dependent K+ (KATP), Ca(2+)-activated K+ (BKCa), and voltage-dependent K+ (KV) currents, and effects of ET-1 on these channels did not differ between SCASMC and LCASMC. From these results, we conclude that Kir channels are expressed at a higher density in SCASMC than in larger arteries and that the Kir channel activity is negatively regulated by the stimulation of ETA-receptors via the PKC pathway.

Atypical gliosis in Müller cells of the slowly degenerating rds mutant mouse retina

Retinal Müller glial cells are known to undergo reactive changes (gliosis) in various retinal diseases. In virtually all cases studied, an upregulation of glial fibrillary acidic protein (GFAP) and a hypertrophy can be observed. Physiological alterations, such as a strong downregulation of inwardly rectifying K + (Kir) currents, were found after retinal detachment (man, rabbit) and after ischemia/reperfusion (rat) but not in more slowly progressing retinal degenerations (Borna Disease Virus-infected rats, RCS rats). This led us to hypothesize that Müller cells respond with ‘typical’ reactive gliosis only to rapid but not to slow retinal degeneration. To test this hypothesis, we studied Müller cells from rds mutant mice ( Prph Rd2 ), which show a retinal degeneration of early onset and slow progression, resulting in a complete loss of photoreceptors after 9–12 months. In Müller cells of rds mice, we found immunoreactivity for GFAP, a marker of gliosis in Müller cells, from postnatal day 21 on, accompanied by a moderately increased membrane capacitance (taken as an indicator of hypertrophy), whereas no change in the expression of the Kir4.1 protein occurred in adult rds mice. We failed to observe significant changes in the membrane resistance and the membrane potential of cells from rds mice from first week after birth until 1 year of age. Current densities were decreased in cells from 3- and 5-week old rds mice. Furthermore, as in control cells from wildtype animals, these cells displayed dominant Kir currents, voltage-dependent Na + currents, and glutamate uptake currents. These data support the idea that in mice as well as previously shown in rats, slow retinal degeneration induces an atypical gliosis of Müller cells.

Contribution of Na+-K+ pump and KIR currents to extracellular pH-dependent changes of contractility in rat superior mesenteric artery

We compared the branches and trunk of rat superior mesenteric artery (SMA) with respect to extracellular pH (pHo)-dependent changes in vascular contractility. Decreases in pHo from 7.8 to 6.4 significantly reduced apparent affinity (pD2) to norepinephrine (NE) and maximal contraction by NE, which were more prominent in larger-diameter arteries. On the other hand, decreases in pHo significantly reduced Ba2+-sensitive K+-induced relaxation (which was evoked by elevation of extracellular K+ concentration from 6 to 12 mM) in the first branch and inhibited inwardly rectifying K+ (K(IR)) currents in cultured smooth muscle cells (SMCs) of SMA. RT-PCR revealed transcripts for Kir2.1 in the SMCs. Real-time PCR analysis revealed 6.1-, 3.3-, and 2.2-fold increases in the Kir2.1 mRNA-to-beta-actin mRNA ratios of SMCs of the third, second, and first branches, respectively, vs. the corresponding relative levels of trunk SMCs. The magnitudes of K+-induced relaxation were significantly greater in smaller-diameter arteries, and there was a strong correlation between the transcript levels of Kir2.1 and K+-induced relaxation. A decrease in pHo reduced ouabain-sensitive K+-induced relaxation and ouabain-induced contraction. A decrease in pHo from 7.4 to 6.4 depolarized membrane potential of the cultured SMCs. From these results, we conclude that an increase in pHo activates K(IR) currents and the Na+ -K+ pump, which then reduces vascular contractility. Inasmuch as K(IR) channel densities are significantly greater in smaller-diameter arteries, the reduction in vascular contractility on increasing pHo is more pronounced in smaller-diameter arteries.

K+ currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

Low affinity block of native and cloned hyperpolarization-activated Ih channels by Ba 2+ ions

Ba 2+ is commonly used to discriminate two classes of ion currents. The classical inward-rectifying K + current, I Kir, is blocked by low millimolar concentrations of Ba 2+, whereas the hyperpolarization-activated cation current, I h, is assumed not to be sensitive to Ba 2+. Here we investigated the effects of Ba 2+ on I h currents recorded from rat hippocampal CA1 pyramidal neurons, and on cloned I h channels composed of either HCN1 or HCN2 subunits transiently expressed in Human Embryonic Kidney (HEK) 293 cells. The results show that low millimolar concentrations of Ba 2+ reduce the maximal I h conductance (IC 50∼3–5 mM) in both CA1 pyramidal neurons and in HEK 293 cells without specificity for HCN1 or HCN2 subunits. In addition, Ba 2+ decreases the rate of activation and increases the rate of deactivation of I h currents. Neither the half-maximal voltage of activation, V h, nor the reversal potential of the I h channels were affected by Ba 2+. The combined results suggest that Ba 2+, at concentrations commonly used to block I Kir currents, also reduces the conductance of I h channels without subunit specificity, and affects the kinetics of I h channel gating.

Altered membrane physiology in Müller glial cells after transient ischemia of the rat retina

Inwardly rectifying K+ (Kir) channels have been implicated in the mediation of retinal K+ homeostasis by Muller glial cells. To assess possible involvement of altered glial K+ channel expression in ischemia-reperfusion injury, transient retinal ischemia was induced in rat eyes. Acutely isolated Muller cells from postischemic retinae displayed a fast downregulation of their Kir currents, which began within 1 day and reached a maximum at 3 days of reperfusion, with a peak decrease to 20% as compared with control. This strong decrease of Kir currents was accompanied by an increase of the incidence of cells which displayed depolarization-evoked fast transient (A-type) K+ currents. While no cell from untreated control rats expressed A-type K+ currents, all cells investigated from 3- and 7-day postischemic retinae displayed such currents. An increased incidence of cells displaying fast transient Na+ currents was observed at 7 days after ischemia. These results suggest a role of altered glial Kir channel expression in postischemic neuronal degeneration via disturbance of retinal K+ siphoning.

Pentobarbital modulates intrinsic and gaba-receptor conductances in thalamocortical inhibition

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 μM, pentobarbital increased input conductance and induced net outward current, I PB , at potentials near action potential threshold. The reversal potential of I PB was −75 mV, implicating K + and other ions. Cs + (3 mM) which inhibits both K + currents and inward rectifier (I h ), completely blocked I PB , whereas the selective I h blocker, ZD-7288 (25 μM), or Ba 2+ (2 mM) which suppresses only K + currents, reduced I PB . Pentobarbital inhibited the I h , consistent with a ZD-7288-induced shift in reversal potential for I PB toward K + equilibrium potential. Pentobarbital increased the inward K + rectifier, I Kir , and leak current, I leak . We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC 50 =53 μM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABA A channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 μM, pentobarbital did not directly activate GABA A receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, I h , I Kir , and I leak , as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Pentobarbital modulates intrinsic and GABA-receptor conductances in thalamocortical inhibition

We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 μM, pentobarbital increased input conductance and induced net outward current, I PB, at potentials near action potential threshold. The reversal potential of I PB was −75 mV, implicating K + and other ions. Cs + (3 mM) which inhibits both K + currents and inward rectifier (I h), completely blocked I PB, whereas the selective I h blocker, ZD-7288 (25 μM), or Ba 2+ (2 mM) which suppresses only K + currents, reduced I PB. Pentobarbital inhibited the I h, consistent with a ZD-7288-induced shift in reversal potential for I PB toward K + equilibrium potential. Pentobarbital increased the inward K + rectifier, I Kir, and leak current, I leak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC 50=53 μM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABA A channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 μM, pentobarbital did not directly activate GABA A receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, I h, I Kir, and I leak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.

Age-related decrease of potassium currents in glial (Müller) cells of the human retina

Background: Age-dependent alterations have been investigated far less in retinal glial cells than in retinal neurons. We investigated age-dependent alterations of inwardly rectifying potassium (Kir) currents in Müller glial cells of the human retina.Methods: Müller cells were isolated immediately post mortem from donors without a reported history of eye disease, and the amplitudes of Kir currents and of currents through high-voltage-activated (HVA) calcium channels were measured by whole-cell patch clamping.Results: The amplitude of the Kir currents was lower in the cells from donors older than 50 years than in the cells of younger donors; the decrease was strongly correlated with the donor's age (p < 0.00 1). The current amplitude in the cells from donors older than 60 years was about 40% lower than the amplitude in the cells from donors younger than 50 years. The amplitude of the HVA currents was greater in the cells from donors older than 55 years than in the cells from younger donors; the increase, up to about 500%, was strongly age-dependent (p < 0.00 1).Interpretation: The age-related decrease in Kir-current amplitude in Muffler cells may reflect the neuron loss in the aged retina. Our findings also indicate that retinal glial cells have enhanced cytoplasmic calcium signals in the course of aging.

Expression of the inwardly rectifying K+ channel Kir2.1 in native bovine corneal endothelial cells.

To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential. RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V(m)) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba(2+). RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a approximately 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K(+) (Kir) current that was dependent on the extracellular K(+) concentration. The Kir current was blocked by external Ba(2+) or Cs(+) in a voltage- and concentration-dependent manner. In 5 mM K(+) Ringer's, the V(m) of cell clusters averaged -40.0 +/- 4.1 mV (n = 14) and in 140 mM K(+) Ringer's it depolarized to -7.4 +/- 1.8 mV. Application of Ba(2+) in 5 mM K(+) Ringer's produced a concentration-dependent depolarization of V(m), with 10 mM Ba(2+) depolarizing V(m) from -53.4 +/- 4.8 mV to -27.8 +/- 6.3 mV (n = 6). Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.

An inwardly rectifying K+ channel in bovine parotid acinar cells: possible involvement of Kir2.1

Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o0.57. The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 µm and 121 mm, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 µm) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.

An inwardly rectifying K+ channel in bovine parotid acinar cells: possible involvement of Kir2.1.

Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.

What are the roles of the many different types of potassium channel expressed in cerebellar granule cells?

Potassium (K) channels have a key role in the regulation of neuronal excitability. Over a hundred different subunits encoding distinct K channel subtypes have been identified so far. A major challenge is to relate these many different channel subunits to the functional K currents observed in native neurons. In this review, we have concentrated on cerebellar granule neurons (CGNs). We have considered each of the three principal super families of K channels in turn, namely, the six transmembrane domain, voltage-gated super family, the two transmembrane domain, inward-rectifier super family and the four transmembrane domain, leak channel super family. For each super family, we have identified the subunits that are expressed in CGNs and related the properties of these expressed channel subunits to the functional currents seen in electrophysiological recordings from these neurons. In some cases, there are strong molecular candidates for proteins underlying observed currents. In other cases the correlation is less clear. We show that at least 26 potassium channel alpha subunits are moderately or strongly expressed in CGNs. Nevertheless, a good empirical model of CGN function has been obtained with just six distinct K conductances. The transient KA current in CGNs, seems due to expression of Kv4.2 channels or Kv4.2/4.3 heteromers, while the KCa current is due to expression of large-conductance slo channels. The G-protein activated KIR current is probably due to heteromeric expression of KIR3.1 and KIR3.2. Perhaps KIR2.2 subunits underlie the KIR current when it is constitutively active. The leak conductance can be attributed to TASK-1 and or TASK-3 channels. With less certainty, the IK-slow current may be due to expression of one or more members of the KCNQ or EAG family. Lastly, the delayed-rectifier Kv current has as many as six different potential contributors from the extensive Kv family of alpha subunits. Since many of these subunits are highly regulated by neurotransmitters, physiological regulators and, often, auxiliary subunits, the resulting electrical properties of CGNs may be highly dynamic and subject to constant fine-tuning.

Growth hormone-releasing peptide-2 reduces inward rectifying K+ currents via a PKA-cAMP-mediated signalling pathway in ovine somatotropes.

Inward-rectifying potassium (Kir) channels are essential for maintaining the resting membrane potential near the K(+) equilibrium and they are responsible for hyperpolarisation-induced K(+) influx. We characterised the Kir current in primary cultured ovine somatotropes and examined the effect of growth hormone-releasing peptide-2 (GHRP-2) on this current and its related intracellular signalling pathways. The Kir current was, in most cases, isolated using nystatin-perforated patch-clamp techniques. In bath solution containing 5 mM K(+), the Kir current was composed of both transient (fast activated) and delayed (slowly activated) components. An increase in the external K(+) concentration from 5 to 25 mM induced an augmentation of approximately 4-fold in the delayed part of the Kir current and both BaCl(2) and CsCl dose-dependently inhibited this current, confirming the presence of the Kir current in ovine somatotropes. Moreover, this specific effect of high K(+) on the Kir current was only observed in the cells that showed positive staining with anti-growth hormone (GH) antibodies, or in GC cells that belong to a rat somatotrope cell line. Application of GHRP-2 (100 nM) reversibly and significantly reduced the Kir current in bath solutions with 5 or 25 mM K(+) in ovine somatotropes. In addition, we found that the reduction in the Kir current mediated by GHRP-2 was totally abolished by the pretreatments with H89 (1 microM) or Rp-cAMP (100 microM) or by intracellular dialysis of a specific protein kinase A (PKA) inhibitory peptide PKI (10 microM). The specific PKC blocker chelerythrine (1 microM) or inhibitory peptide PKC(19-36) (10 microM) did not show any effects on the GHRP-2-induced decrease in the Kir current. These results suggest that the inhibition of Kir current through PKA-cAMP pathways may play an integral role in GHRP-2-induced depolarisation and GH release in ovine somatotropes.

Inhibition of inwardly rectifying K(+) channels by cGMP in pulmonary vascular endothelial cells.

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca(2+) concentration. Membrane-permeable analogs of guanosine 3',5'-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential (E(m)), which influences the electrochemical gradient for Ca(2+) influx, is regulated by K(+) channels, we investigated the effect of 8-bromo-cGMP on E(m) and inwardly rectifying K(+) (K(IR)) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to -50 mV that were abolished by application of 10 microM Ba(2+), consistent with K(IR) current. Ba(2+) also depolarized both cell types. 8-Bromo-cGMP (10(-3) M) depolarized BPAEC and BMVEC and inhibited K(IR) current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of K(IR) current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca(2+) entry.