Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

Effects of calyculin A on amylase release in streptolysin-O permeabilized acinar cells.

The effects of the phosphatase inhibitors calyculin A and okadaic acid on amylase release from streptolysin-O permeabilized rat pancreatic acini were investigated. Both agents induced similar biphasic effects with moderate potentiation of calcium-stimulated amylase release at medium and strong inhibition at higher concentrations. Calyculin A was thirty times more potent than okadaic acid and at 100 nM totally inhibited calcium-induced amylase release while 3 microM okadaic acid reduced amylase release by 78%. 100nM calyculin A also completely inhibited GTP gamma S-potentiated amylase release and partially inhibited phorbol ester potentiated secretion. The data indicate that inhibition of a serine/threonine phosphatase, probably a type 1 phosphatase, leads to inhibition of calcium-induced amylase release in permeabilized pancreatic acini.     

Development of different electrophysiological mechanisms for muscarinic inhibition of atria and ventricles

The negative inotropic effect of acetylcholine (ACh) in atrial muscle can be accounted for by a decrease of a voltage- and time-dependent slow inward current (Isi) carried by Ca2+/Na+ and an increase of outward time-dependent current carried by K+ (IK1) through inwardly rectifying channels. The negative inotropic effect of ACh in ventricular muscle is associated with a reduction of Isi; there is no important effect of ACh on IK1 in ventricular muscle. Because atrial and ventricular muscles display IK1 that is sensitive to Ba2+ and have similar numbers of muscarinic receptor sites, it is concluded that ventricular muscle lacks a metabolic link between the muscarinic receptor and inwardly rectifying K+ channels. Although there is much evidence for cyclic nucleotides as the mediator between muscarinic receptors and Isi channels, cyclic nucleotides do not seem to connect these receptors with inwardly rectifying K+ channels. According to this hypothesis, identification of a metabolic link between muscarinic receptors and IK1 channels should be demonstrable in atrial but not ventricular muscle.     

Are Redox Reactions Involved in Regulation of K+ Channels in the Plasma Membrane of Limnobium stoloniferum Root Hairs?

The effects of the impermeant electron acceptor hexacyanoferrate III (HCF III) and the potassium channel blocker tetraethylam-monium (TEA) on the current-voltage relationship and electrical potential across the plasma membrane of Limnobium stoloniferum root hairs was investigated using a modified sucrose gap technique. One millimolar HCF III immediately and reversibly depolarized the membrane by 27 mV, whereas the effect on the trans-membrane current was markedly delayed. After 6 min of treatment with this electron acceptor, outwardly rectifying current was inhibited by 50%, whereas the inwardly rectifying current was activated approximately 3-fold. Ten millimolar TEA blocked both outward (65%) and inward (52%) currents. Differential TEA-sensitive current was shown to be blocked (55%) by HCF III at -20 mV and was shown to be stimulated (230%) by this electron acceptor at -200 mV. The inward current at -200 mV was eliminated in the absence of K+ or after addition of 10 mM Cs+ and was not affected by addition of either 10mM Na+ or Li+, independent of the presence of HCF III. The addition of any alkali cation to the external medium decreased the outward current both in the presence and in the absence of HCF III. The membrane depolarization evoked by HCF III did not correlate with the corresponding modification of the inward current. HCF III is proposed to activate inwardly rectifying potassium channels and to inactivate outwardly rectifying potassium channels. It is concluded that the plasma membrane depolarization did not result from modulation of the potassium channels by HCF III and may originate from trans-plasma membrane electron transfer.     

Inward rectification by hyperpolarization-activated Na current in the marine ciliate Euplotes vannus

Summary The ionic mechanisms underlying inward or anomalous rectification have been studied in the marine hypotrichous ciliate Euplotes vannus. Inward-current pulses of moderate amplitude elicited time-dependent rectification that started from a hyperpolarization peak and was expressed as a depolarizing sag towards rest. Voltage-clamp analysis showed that this depolarization is caused by the activation of a complex inward current that does not inactivate with time. The current is carried by a major Na and a minor K component. The Na-current component has been identified by its concentration-dependent reduction in low extra-cellular Na solutions and the capability of Li²⁺ as Na substitute to carry the current, though with a slightly reduced amplitude. The K-current component has been isolated from the total current after the replacement of Na²⁺ within the experimental solution. It was blocked in media that contained 10 mmol/liter TEA, a well-known blocker for K inwardly rectifying currents. TEA was only effective at membrane potentials close to or negative to the potassium equilibrium potential. The inward current was reduced after the injection of the Ca chelator EGTA into the cell. Also the elimination of the ciliary membrane, by deciliating cells with ethanol, reduced the amplitude of the inwardly rectifying currents. Both experiments indicate a regulatory function of Ca²²⁺ in inward rectification.The author is grateful to Harald Mikoleit for technical assistance and preparing the figures and to Prof. W. Lueken for his critical comments. This work was supported by Deutsche Forschungsgemeinschaft, SFB 171, C7.     

G-protein activators induce a potassium conductance in murine macrophages

The whole-cell patch clamp technique was used to test whether intracellular application of G-protein activators affect ionic currents in murine macrophages. Both the J774.1 macrophage-like cell line and primary bone marrow derived macrophages were used. Cells were bathed in Na Hanks' solution and intracellularly dialyzed (via the patch pipette) with K Hanks (145 mM KCl, < 100 nM Ca) plus or minus the G-protein activators GTP gamma S (10 microM), GppNHp (10 microM), or AIF4- (200 microM AlCl3 + 5 mM KF). In the absence of G-protein activators, only two K currents, an inwardly rectifying K current (Kir) and an outward, inactivating K current (Ko) were observed. In the presence of protein activators, two effects were observed: (i) the Kir conductance, which is stable for up to 30 min under control conditions, decayed twice as fast and (ii) an outwardly rectifying, noninactivating current appeared. The induced outward current appeared < 2 min after attaining the whole-cell patch clamp configuration. The current could be distinguished from the Kir and Ko currents on the basis of its direction of rectification (outward), barium sensitivity (> 1 mM), and kinetics (no time-dependent inactivation). Intracellular application of GTP (500 microM), GDP (500 microM), cAMP (100 microM + 0.5 mM ATP), or IP3 (20 microM) did not induce the current; 100 microM ATP gamma S activated a half-maximal amount of current. Induction of outward current by 10 microM GTP gamma S could be prevented by pre-exposing cells to pertussis toxin but not cholera toxin. This current is K selective since (i) its induction was accompanied by hyperpolarization of the cell toward EK, even after Kir had "washed out", (ii) it was present after > 90% of both intracellular and extracellular Cl were replaced by isethionate, and (iii) the induced outward conductance was absent when Ki was completely replaced by Cs, and was reduced by approximately 1/3 when [K]i was reduced by 1/3. Quinidine (1 mM) and 4-aminopyridine (10 mM) inhibited the current, but apamin (1 microM) and charybdotoxin (1 microM) did not.     

Inhibition of Ca2+-activated and voltage-dependent K+ currents by 2-mercaptophenyl-1,4-naphthoquinone in pituitary GH3 cells: contribution to its antiproliferative effect

Quinones have been shown to possess antineoplastic activity; however, their effects on ionic currents remain unclear. The effects of 2-mercaptophenyl-1,4-naphthoquinone (2-MPNQ), menadione (MD) and 1,4-naphthoquinone (1,4 NQ) on cell proliferation and ionic currents in pituitary GH3 lactotrophs were investigated in this study. 2-MPNQ was more potent than menadione or 1,4-naphthoquinone in inhibiting the growth of GH3 cells. 2-MPNQ decreased cell proliferation in a concentration-dependent manner with an IC50 value of 3 microM. In whole-cell recording experiments, 2-MPNQ reversibly caused an inhibition of Ca2+-activated K+ current (I(K(Ca)) in a concentration-dependent manner. The IC50 value for 2-MPNQ-induced inhibition of I(K(Ca)) was 7 microM. In the inside-out configuration of single channel recording, 2-MPNQ (30 microM) applied intracellularly suppressed the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels but did not modify single channel conductance. Menadione (30 microM) had no effect on the channel activity, whereas 1,4-naphthoquinone (30 microM) suppressed it by about 26%. Both 2-MPNQ and thimerosal suppressed the dithiothreitol-stimulated channel activity. 2-MPNQ also blocked voltage-dependent K+ currents, but it produced a slight reduction of L-type Ca2+ inward current. However, unlike E-4031, 2-MPNQ (30 microM) did not suppress inwardly rectifying K+ current present in GH3 cells. Under the current clamp configuration, the presence of 2-MPNQ (30 microM) depolarized the cells, and increased the frequency and duration of spontaneous action potentials. The 2-MPNQ-mediated inhibition of K+ currents would affect hormone secretion and cell excitability. The blockade of these ionic channels by 2-MPNQ may partly explain its inhibitory effect on the proliferation of GH3 cells.     

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.     

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.     

Two ATP-activated conductances in bullfrog atrial cells

Currents activated by extracellular ATP were studied in single voltage-clamped bullfrog atrial cells. Rapid application of ATP elicited currents carried through two different conductance pathways: a rapidly desensitizing conductance reversing near -10 mV, and a maintained, inwardly rectifying conductance reversing near -85 mV. ATP activated the desensitizing component of current with a K 1/2 of approximately 50 microM and the maintained component with a K 1/2 of approximately 10 microM. Both types of current were activated by ATP but not by adenosine, AMP, or ADP. The desensitizing current was selectively inhibited by alpha, beta-methylene ATP, and the maintained, inwardly rectifying current was selectively suppressed by extracellular Cs. The desensitizing component of current was greatly reduced when extracellular Na was replaced by N-methylglucamine, but was slightly augmented when Na was replaced by Cs. GTP, ITP, and UTP were all ineffective in activating the desensitizing current, and of a variety of ATP analogues, only ATP-gamma-S was effective. Addition of EGTA or BAPTA to the intracellular solution did not obviously affect the desensitizing current. Fluctuation analysis of currents through the desensitizing conductance suggested that current is carried through ionic channels with a small (less than pS) unitary conductance.     

[K+] dependence of polyamine-induced rectification in inward rectifier potassium channels (IRK1, Kir2.1)

The effects of permeant (K+) ions on polyamine (PA)-induced rectification of cloned strong inwardly rectifying channels (IRK1, Kir2.1) expressed in Xenopus oocytes were examined using patch-clamp techniques. The kinetics of PA-induced rectification depend strongly on external, but not internal, K+ concentration. Increasing external [K+] speeds up "activation" kinetics and shifts rectification to more positive membrane potentials. The shift of rectification is directly proportional to the shift in the K+ reversal potential (EK) with slope factors +0.62, +0.81, and +0.91 for 1 mM putrescine (Put), 100 microM spermidine and 20 microM spermine (Spm), respectively. The time constant of current activation, resulting from unblock of Spm, also shifts directly in proportion to EK with slope factor +1.1. Increasing internal [K+] slows down activation kinetics and has a much weaker relieving effect on block by PA: Spm-induced rectification and time constant of activation (Spm unblock) shift directly in proportion to the corresponding change in EK with slope factors -0.15 and +0.31, respectively, for 20 microM Spm. The speed up of activation kinetics caused by increase of external [K+] cannot be reversed by equal increase of internal [K+]. The data are consistent with the hypothesis that the conduction pathway of strong inward rectifiers is a long and narrow pore with multiple binding sites for PA and K+.     

Inhibition, by an amphiphilic substance, niflumic acid, of the inward rectification of the crustacean muscle fiber

Agents such as TEA+ or CS+ ions, these last ions instead of K+ ions in poor K extracellular solution, known to reduce or abolish the inwardly rectifying channel in many preparations produced no effect in crayfish muscle membrane By contrast, poor Cl extracellular solution (Cl- ions were replaced by CH3OSO3- ions) blocked the inward current activated by hyperpolarizing pulses and produced an increase of the resting potential. Niflumic acid is a agent which inhibited the inward going rectification of the crayfish muscle membrane. Apparent dissociation constant of niflumic acid with membrane sites was equal to about 6 X 10(-8) M; this value corresponds to that given by Cousin & Motais (1979) concerning translocation of Cl- ions in the membrane of red cells. Activation of the inward going rectification in the crayfish membrane is responsible of an inward current carried by Cl- ions.     

Ca(2+)-insensitive modulation of a K+ conductance by inositol polyphosphates

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.     

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Block of inward rectification by intracellular H+ in immature oocytes of the starfish Mediaster aequalis

Intracellular pH was recorded in immature starfish oocytes using pH- sensitive microelectrodes, and inwardly rectifying potassium currents were measured under voltage clamp. When the intracellular pH was lowered using acetate-buffered artificial sea water from the normal value of 7.09 to 5.9, inward rectification was completely blocked. The relationship between inward rectification and internal pH between 7.09 and 5.9 could be fit by a titration curve for the binding of three H ions to a site with a pK of 6.26 to block the channel. The H+ block showed no voltage dependence, and the activation kinetics of the inwardly rectifying currents were not affected by the changes in internal pH.     

Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.     

G protein-mediated Ca-sensitization of CPI-17 phosphorylation in arterial smooth muscle

CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain phosphatase in smooth muscle and plays an essential role in agonist-induced contraction. To elucidate the in situ mechanism for G protein-mediated Ca²⁺-sensitization of CPI-17 phosphorylation, α-toxin-permeabilized arterial smooth muscle strips were used to monitor both force development and CPI-17 phosphorylation in response to GTPγS with varying Ca²⁺ concentrations. CPI-17 phosphorylation increased at unphysiologically high Ca²⁺ levels of pCa ? 6. GTPγS markedly enhanced the Ca²⁺ sensitivity of CPI-17 steady-state phosphorylation but had no enhancing effect under Ca²⁺-free conditions, while the potent PKC activator PDBu increased CPI-17 phosphorylation regardless of Ca²⁺ concentration. CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A, a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with time even under Ca²⁺-free conditions. Furthermore, as Ca²⁺ concentration increased, so did CPI-17 phosphorylation rate. GTPγS markedly enhanced the rate of phosphorylation of CPI-17 at a given Ca²⁺. In the absence of calyculin A, either steady-state phosphorylation of CPI-17 under Ca²⁺-free conditions in the presence of GTPγS or at pCa 6.7 in the absence of GTPγS was negligible, suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion, cooperative increases in Ca²⁺ and G protein activation are required for a significant activation of total kinases that phosphorylate CPI-17, which together overcome CPI-17 phosphatase activity and effectively increase the Ca²⁺ sensitivity of CPI-17 phosphorylation and smooth muscle contraction.     

Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin

M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex.