Stephen J. Marsh

Molecular correlates of the M‐current in cultured rat hippocampal neurons

M‐type K+ currents (IK(M)) play a key role in regulating neuronal excitability. In sympathetic neurons, M‐channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K+ channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of IK(M) in hippocampal pyramidal neurons cultured from 5‐ to 7‐day‐old rats. RT‐PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single‐cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M‐channel blocker, fully inhibited IK(M) with an IC50 of 3.6 ± 1.5 μM. In 70 % of these cells, TEA fully suppressed IK(M) with an IC50 of 0.7 ± 0.1 mm. In the remaining cells, TEA maximally reduced IK(M) by only 59.7 ± 5.2 % with an IC50 of 1.4 ± 0.3 mm; residual IK(M) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to IK(M) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.

Relationship between Membrane Phosphatidylinositol-4,5-Bisphosphate and Receptor-Mediated Inhibition of Native Neuronal M Channels

The relationship between receptor-induced membrane phosphatidylinositol-4′5′-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCδ-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCδ-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCδ-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 μm (95% confidence limit; 192-381 μm), rising to 693 μm (417-1153 μm) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.

CALCIUM-ENTRY THROUGH NICOTINIC RECEPTOR CHANNELS AND CALCIUM CHANNELS IN CULTURED RAT SUPERIOR CERVICAL-GANGLION CELLS

1. Patch‐clamp techniques in conjunction with indo‐1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage‐clamp conditions, pressure application of the nicotinic agonist DMPP (1,1‐dimethyl‐4‐phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/‐ 26 nM at a clamp potential of ‐60 mV. This was accompanied by an inward current of ‐4.53 +/‐ 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP‐induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM‐10 microM) but were unaffected by alpha‐bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from ‐80 to ‐40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine‐sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage‐dependent calcium current. On average at ‐60 mV, the M/q ratio of this highly calcium‐selective permeability was 1961 mM nC‐1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant‐field theory, ion‐substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

The plasma membrane calcium-ATPase as a major mechanism for intracellular calcium regulation in neurones from the rat superior cervical ganglion

Patch‐clamp recording combined with indo‐1 measurement of free intracellular calcium concentration ([Ca2+]i) was used to determine the homeostatic systems involved in the maintenance of resting [Ca2+]i and in the clearance of Ca2+ transients following activation of voltage‐gated Ca2+ channels in neurones cultured from rat superior cervical ganglion (SCG). The Ca2+ binding ratio was estimated to be ∼500 at 100 nM, decreasing to ∼250 at [Ca2+]i≈ 1 μM, and to involve at least two buffering systems with different affinities for Ca2+. Removal of extracellular Ca2+ led to a decrease in [Ca2+]i that was mimicked by the addition of La3+, and was more pronounced after inhibition of the endoplasmic reticulum Ca2+ uptake system (SERCA). Inhibition of the plasma membrane Ca2+ pump (PMCA) by extracellular alkalinisation (pH 9) or intracellular carboxyeosin both increased resting [Ca2+]i and prolonged the recovery of Ca2+ transients at peak [Ca2+]i⩽ 500 nM. For [Ca2+]i loads > 500 nM, recovery showed an additional plateau phase that was abolished in m‐chlorophenylhydrazone (CCCP) or on omitting intracellular Na+. Inhibition of the plasma membrane Na+ ‐Ca2+ exchanger (NCX) and of SERCA had a small but significant additional effect on the rate of decay of these larger Ca2+ transients. In conclusion, resting [Ca2+]i is maintained by passive Ca2+ influx and regulated by a large Ca2+ buffering system, Ca2+ extrusion via a PMCA and Ca2+ transport from the intracellular stores. PMCA is also the principal Ca2+ extrusion system at low Ca2+ loads, with additional participation of the NCX and intracellular organelles at high [Ca2+]i.

Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

1. The M‐like current IK(M,ng) in differentiated NG108‐15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight‐seal, whole‐cell patch‐clamp recording. 2. When calculated from steady‐state current‐voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half‐activation voltage Vo = ‐44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e‐fold increase in conductance. In 12 mM [K+] Vo = ‐38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e‐fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady‐state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo‐1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)‐free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9‐amino‐1,2,3,4‐tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4‐aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1‐10 microM) or angiotensin II (1‐10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5‐hydroxytryptamine (10 microM), Met‐enkephalin (1 microM), glycine (100 microM), gamma‐aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of M(Kv7.2/7.3) channels in neurons by PIP2 and products of PIP2 hydrolysis: significance for receptor-mediated inhibition

M‐channels are voltage‐gated K+ channels that regulate the excitability of many neurons. They are composed of Kv7 (KCNQ) family subunits, usually Kv7.2 + Kv7.3. Native M‐channels and expressed Kv7.2 + 7.3 channels are inhibited by stimulating Gq/11‐coupled receptors – prototypically the M1 muscarinic acetylcholine receptor (M1‐mAChR). The channels require membrane phosphatidylinositol‐4,5‐bisphosphate (PIP2) to open and the effects of mAChR stimulation result primarily from the reduction in membrane PIP2 levels following Gq/phospholipase C‐catalysed PIP2 hydrolysis. However, in sympathetic neurons, M‐current inhibition by bradykinin appears to be mediated through the release and action of intracellular Ca2+ by inositol‐1,4,5‐trisphosphate (IP3), a product of PIP2 hydrolysis, rather than by PIP2 depletion. We have therefore compared the effects of bradykinin and oxotremorine‐M (a muscarinic agonist) on membrane PIP2 in sympathetic neurons using a fluorescently tagged mutated C‐domain of the PIP2 binding probe, ‘tubby’. In concentrations producing equal M‐current inhibition, bradykinin produced about one‐quarter of the reduction in PIP2 produced by oxotremorine‐M, but equal reduction when PIP2 synthesis was blocked with wortmannin. Likewise, wortmannin restored bradykinin‐induced M‐current inhibition when Ca2+ release was prevented with thapsigargin. Thus, inhibition by bradykinin can use product (IP3/Ca2+)‐dependent or substrate (PIP2) dependent mechanisms, depending on Ca2+ availability and PIP2 synthesis rates.

On the mechanism of M‐current inhibition by muscarinic m1 receptors in DNA‐transfected rodent neuroblastoma x glioma cells.

1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108‐15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)‐dependent K+ conductance, producing an outward current, and it inhibits a voltage‐dependent K+ conductance (the M conductance), thus diminishing the M‐type voltage‐dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo‐1 using whole‐cell patch‐clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/‐ 3.2% (n = 23). Following pressure‐application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G‐protein was tested by adding 0.5 mM GTP‐gamma‐S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half‐times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml‐1 pertussis toxin or on adding up to 10 mM GDP‐beta‐S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5‐trisphosphate (InsP3) and inositol 1,3,4,5‐tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh‐induced outward current and/or intracellular Ca2+ transient but did not block ACh‐induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach‐induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic M1 receptors activate phosphoinositide turnover and Ca2+ mobilisation in rat sympathetic neurones, but this signalling pathway does not mediate M‐current inhibition

1 The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M‐current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. 2 Phosphoinositide‐specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]‐cytidine monophosphate phosphatidate (CMP‐PA) after incubation with [3H]‐cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo‐M) stimulated PLC in a dose‐dependent manner with an EC50 of approximately 3.5 μm. 3 The concentration‐response curve for oxo‐M was shifted to the right by a factor of about 10 by pirenzepine (100 nm), suggesting a pKB (—log of the apparent dissociation constant) of 7.9 ± 0.4, while himbacine (1 μm) shifted the curve by a factor of about 13 (pKB∼7.1 ± 0.6). This indicates involvement of the M1 muscarinic receptor in this response. 4 The accumulation of CMP‐PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. 5 The ability of oxo‐M to release Ca2+ from inositol(1,4,5)trisphosphate (InsP3)‐sensitive stores was determined by fura‐2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo‐M failed to evoke intracellular Ca2+ (Ca2+i) mobilisation in SCG neurones voltage clamped at −60 mV, but produced a significant Ca2+i rise (67 ± 15 nm, n= 9) in cells voltage clamped at −25 mV. 6 Thapsigargin (0.5–1 μm) caused a 70% inhibition of the oxo‐M‐induced Ca2+i increase, indicating its intracellular origin, while oxo‐M‐induced inhibition of M‐current in the same cells was unaffected by thapsigargin. 7 Our results do not support the involvement of InsP3‐sensitive calcium mobilisation in M‐current inhibition.

Probing the Regulation of M (Kv7) Potassium Channels in Intact Neurons with Membrane-Targeted Peptides

M-type (Kv7) potassium channels are closed by Gq/11 G-protein-coupled receptors. Several membrane- or channel-associated molecules have been suggested to contribute to this effect, including depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) and activation of Ca2+/calmodulin and protein kinase C. To facilitate further study of these pathways in intact neurons, we have devised novel membrane-targeted probes that can be applied from the outside of the neuron, by attaching a palmitoyl group to site-directed peptides (“palpeptides”) (cf. Covic et al., 2002a,b). A palpeptide incorporating the 10-residue C terminus of Gαq/11 reduced Gq/11-mediated M-current inhibition in sympathetic neurons by the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M but not Go-mediated inhibition of the N-type Ca2+ current by norepinephrine. Instead, the latter was inhibited by the corresponding Go palpeptide. A PIP2 palpeptide, based on the putative PIP2 binding domain of the Kv7.2 channel, inhibited M current (IC50 = ∼1.5 μm) and enhanced inhibition by oxotremorine-M. Inhibition could not be attributed to activation of mAChRs, calcium influx, or block of M channels but was antagonized by intracellular diC8-PIP2 (dioctanoyl-phosphatidylinositol-4,5-bisphosphate), suggesting that it disrupted PIP2–M channel gating. A fluorescently tagged PIP2 palpeptide was highly targeted to the plasma membrane but did not accumulate in the cytoplasm. We suggest that these palpeptides are anchored in the plasma membrane via the palmitoyl group, such that the peptide moiety can interact with target molecules on the inner face of the membrane. The G-protein-replicating palpeptides were sequence specific and probably compete with the receptor for the cognate G-protein. The PIP2 palpeptide was not sequence specific so probably interacts electrostatically with anionic PIP2 head groups.

PIP2-dependent inhibition of M-type (Kv7.2/7.3) potassium channels: direct on-line assessment of PIP2 depletion by Gq-coupled receptors in single living neurons

The open state of M(Kv7.2/7.3) potassium channels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). They can be closed on stimulating receptors that induce PI(4,5)P2 hydrolysis. In sympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has been attributed to depletion of PI(4,5)P2, whereas closure by bradykinin B2-receptors (B2-BKRs) appears to result from formation of IP3 and release of Ca2+, implying that BKR stimulation does not deplete PI(4,5)P2. We have used a fluorescently tagged PI(4,5)P2-binding construct, the C-domain of the protein tubby, mutated to increase sensitivity to PI(4,5)P2 changes (tubby-R332H-cYFP), to provide an on-line read-out of PI(4,5)P2 changes in single living sympathetic neurons after receptor stimulation. We find that the mAChR agonist, oxotremorine-M (oxo-M), produces a near-complete translocation of tubby-R332H-cYFP into the cytoplasm, whereas bradykinin (BK) produced about one third as much translocation. However, translocation by BK was increased to equal that produced by oxo-M when synthesis of PI(4,5)P2 was inhibited by wortmannin. Further, wortmannin ‘rescued’ M-current inhibition by BK after Ca2+-dependent inhibition was reduced by thapsigargin. These results provide the first direct support for the view that BK accelerates PI(4,5)P2 synthesis in these neurons, and show that the mechanism of BKR-induced inhibition can be switched from Ca2+ dependent to PI(4,5)P2 dependent when PI(4,5)P2 synthesis is inhibited.