Overexpression of Adhesion Molecule L1 in NG108-15 Neuroblastoma × Glioma Hybrid Cells Enhances Dibutyryl Cyclic AMP-Induced Neurite Outgrowth and Functional Synapse Formation with Myotubes

Abstract: The role of adhesion molecule L1 in synapse formation was examined by transient transfection of L1 cDNA in neuroblastoma × glioma hybrid NG108-15 cells. L1 overexpression was found in ∼50% of the transfected NG108-15 cell population. Neurite outgrowth induced by 0.25 m M dibutyryl cyclic AMP (cAMP) was much greater in L1-transfected NG108-15 cells than that in nontransfected and mock-transfected cells. The proportion of cells with neurites and the number of neurites per cells were increased in L1-transfected cells after 2 days of dibutyryl cAMP treatment. The proportion of cells with branched neurites and the average length of neurites were higher at day 4. A significantly higher rate of synapse formation with myotubes was apparent in the late phase of coculture (days 4–7) in L1-transfected cells than in control cells. The miniature end-plate potential frequency in myotubes was the same for the three types of NG108-15 cells. These results show that overexpression of L1 in NG108-15 cells facilitates synaptic connections by enhancing branching and elongation of neurites induced with dibutyryl cAMP, rather than by increasing probability of acetylcholine release.     

Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.     

Characterization of heteromultimeric G protein-coupled inwardly rectifying potassium channels of the tunicate tadpole with a unique pore property

Two cDNAs that encode the G protein-coupled inwardly rectifying K(+) channel (GIRK, Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly rectifying K(+) channel-A and -B; TuGIRK-A and -B) have been isolated. The deduced amino acid sequences showed approximately 60% identity with the mammalian Kir3 family. Detected by whole mount in situ hybridization, both TuGIRK-A and -B were expressed similarly in the neural cells of the head and neck region from the tail bud stage to the young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein beta(1)/gamma(2) subunits (Gbetagamma) in Xenopus oocytes, an inwardly rectifying K(+) current was expressed. In contrast, coinjection of TuGIRK-B with Gbetagamma did not express any current. When both TuGIRK-A and -B were coexpressed together with Gbetagamma, an inwardly rectifying K(+) current was also detected. The properties of this current clearly differed from those of TuGIRK-A current, since it displayed a characteristic decline of the macroscopic conductance at strongly hyperpolarized potentials. TuGIRK-A/B current also differed from TuGIRK-A current in terms of the lower sensitivity to the Ba(2+) block, the higher sensitivity to the Cs(+) block, and the smaller single channel conductance. Taken together, we concluded that TuGIRK-A and -B form functional heteromultimeric G protein-coupled inwardly rectifying K(+) channels in the neural cells of the tunicate tadpole. By introducing a mutation of Lys(161) to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a higher sensitivity to the Ba(2+) block and a slightly lower sensitivity to the Cs(+) block, and the decrease in the macroscopic conductance at hyperpolarized potentials was no longer observed. Thus, the differences in the electrophysiological properties between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least partly, due to the presence of Lys(161) at the external mouth of the pore of the TuGIRK-B subunit.     

Genetic and functional linkage of Kir5.1 and Kir2.1 channel subunits

We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K(+) channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1-24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.     

Tyrosine phosphorylation of K(ir)3.1 in spinal cord is induced by acute inflammation, chronic neuropathic pain, and behavioral stress

Tyrosine phosphorylation is an important means of regulating ion channel function. Our previous gene expression studies using the Xenopus laevis oocyte system suggested that tyrosine phosphorylation of G-protein-gated inwardly rectifying potassium channels (K(ir)3 or GIRK) suppressed basal channel conductance and accelerated channel deactivation. To assess whether similar mechanisms regulate K(ir)3 function in mammalian cells, we developed and characterized a phosphoselective antibody recognizing K(ir)3.1 phosphorylated at tyrosine 12 in the N-terminal domain and then probed for evidence of K(ir)3.1 phosphorylation in cultured mammalian cells and spinal cord. The antibody was found to discriminate between the phospho-Tyr(12) of K(ir)3.1 and the native state in transfected cell lines and in primary cultures of mouse atria. Following either mouse hindpaw formalin injection or sciatic nerve ligation, pY12-K(ir)3.1 immunoreactivity was enhanced unilaterally in the superficial layers of the spinal cord dorsal horn, regions previously described as expressing K(ir)3.1 channels. Mice lacking K 3.1 following targeted gene disruption did not show specific pY12-K(ir)3.1 immunoreactivity after sciatic nerve ligation. Further, mice exposed to repeatedly forced swim stress showed bilateral enhancement in pY12-K(ir)3.1 in the dorsal horn. This study provides evidence that K(ir)3 tyrosine phosphorylation occurred during acute and chronic inflammatory pain and under behavioral stress. The reduction in K(ir)3 channel activity is predicted to enhance neuronal excitability under physiologically relevant conditions and may mediate a component of the adaptive physiological response.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

A recombinant inwardly rectifying potassium channel coupled to GTP- binding proteins

GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.     

Expression cloning of KCRF, a potassium channel regulatory factor

By functional coexpression screening of a rat cDNA library in Xenopus oocytes, we have cloned a protein (KCRF: K Channel Regulatory Factor) that reduces currents of several K(+) channels: G protein-activated GIRK1/4 (K(ir)3.1/K(ir)3.4), inward rectifier IRK1 (K(ir)2.1), and voltage-dependent K(V)1.1/K(V)beta1.1. KCRF did not modulate two other K(+) channels: ROMK1 (K(ir)1.1) and GIRK1/2 (K(ir)3.1/K(ir)3.2) and the voltage-dependent L-type Ca(2+) channels. Western blot analysis showed that KCRF is ubiquitous in rat tissues. Biochemical and electrophysiological experiments revealed that coexpression of KCRF causes a decrease in the level of expression of IRK1 and K(V)1.1/K(V)beta1.1 proteins in the oocytes.     

Plant K+ channel alpha-subunits assemble indiscriminately.

In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression.     

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.     

Regulation of ROMK by extracellular cations

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (K(ir)1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10 mM increased whole-cell outward conductance by a factor of 3.4 +/- 0.4 in 15 min and by a factor of 5.7 +/- 0.9 in 30 min (n = 22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5 +/- 0.5 over a period of 40 min (n = 15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10 mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4 +/- 0.8 pS to 11.1 +/- 0.8 pS (10 mM K, n = 8) or 11.7 +/- 1.2 pS (Cs, n = 8). There was no effect of either 10 mM K or cesium on the high open probability (P(o) = 0.97 +/- 0.01; n = 12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50 mM K. In cell-attached patches, exposure to 50 mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50 mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (K(ir)2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10 mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.     

Expression of an inwardly rectifying K channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Long chain CoA esters as competitive antagonists of phosphatidylinositol 4,5-bisphosphate activation in Kir channels

Long chain fatty acid esters of coenzyme A (LC-CoA) are potent activators of ATP-sensitive (K(ATP)) channels, and elevated levels have been implicated in the pathophysiology of type 2 diabetes. This stimulatory effect is thought to involve a mechanism similar to phosphatidylinositol 4,5-bisphosphate (PIP2), which activates all known inwardly rectifying potassium (Kir) channels. However, the effect of LC-CoA on other Kir channels has not been well characterized. In this study, we show that in contrast to their stimulatory effect on K(ATP) channels, LC-CoA (e.g. oleoyl-CoA) potently and reversibly inhibits all other Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir7.1). We also demonstrate that the inhibitory potency of the LC-CoA increases with the chain length of the fatty acid chain, while both its activatory and inhibitory effects critically depend on the presence of the 3'-ribose phosphate on the CoA group. Biochemical studies also demonstrate that PIP2 and LC-CoA bind with similar affinity to the C-terminal domains of Kir2.1 and Kir6.2 and that PIP2 binding can be competitively antagonized by LC-CoA, suggesting that the mechanism of LC-CoA inhibition involves displacement of PIP2. Furthermore, we demonstrate that in contrast to its stimulatory effect on K(ATP) channels, phosphatidylinositol 3,4-bisphosphate has an inhibitory effect on Kir1.1 and Kir2.1. These results demonstrate a bi-directional modulation of Kir channel activity by LC-CoA and phosphoinositides and suggest that changes in fatty acid metabolism (e.g. LC-CoA production) could have profound and widespread effects on cellular electrical activity.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.