A. A. Selyanko

Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors

KCNQ1‐4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole‐cell perforated patch technique and cell‐attached patch recording. Stimulation of M1 receptors by 10 μm oxotremorine‐M (Oxo‐M) strongly reduced (to 0–10%) currents produced by KCNQ1‐4 subunits expressed individually and also those produced by KCNQ2+KCNQ3 and KCNQ1+KCNE1 heteromers, which are thought to generate neuronal M‐currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. The activity of KCNQ2+KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell‐attached pipettes was strongly and reversibly reduced by Oxo‐M applied to the extra‐patch membrane. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2+KCNQ3 channels, like that of native M‐channels, requires a diffusible second messenger.

Differential tetraethylammonium sensitivity of KCNQ1–4 potassium channels

In Shaker‐group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild‐type KCNQ1–4 channels and heteromeric KCNQ2/3 channels incorporating either wild‐type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, >30; KCNQ4, 3.0; KCNQ2+KCNQ3, 3.8; and KCNQ2+KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.

Muscarinic mechanisms in nerve cells

The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.

Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells

1 The single channel properties of KCNQ2/KCNQ3 channels underlying neuronal voltage‐dependent M‐type potassium currents were studied in cell‐attached patches from transfected Chinese hamster ovary (CHO) cells. Macroscopic currents produced by homo‐ and heteromeric KCNQ2/KCNQ3 channels were measured using the perforated‐patch whole‐cell technique. 2 Compared with heteromeric KCNQ2 + KCNQ3 channels, homomeric KCNQ2 channels had lower slope conductance (9.0 ± 0.3 and 5.8 ± 0.3 pS, respectively) and open probability at 0 mV (0.30 ± 0.07 and 0.15 ± 0.03, respectively), consistent with their 3.8‐fold smaller macroscopic currents. By contrast, homomeric KCNQ3 channels had the same slope conductance (9.0 ± 1.1 pS) as KCNQ2 + KCNQ3 channels, and higher open probability (0.59 ± 0.11), inconsistent with their 12.7‐fold smaller macroscopic currents. Thus, KCNQ2 and KCNQ3 subunits may play different roles in the expression of M‐type currents, with KCNQ2 ensuring surface expression of underlying channels and KCNQ3 modifying their function. 2 Both in homo‐ and heteromeric KCNQ2/KCNQ3 channels the shut time distributions were fitted with three, and the open time distributions with two, exponential components. By measuring these and other parameters (e.g. conductance and open probability) KCNQ2/ KCNQ3 channels can be shown to resemble previously characterised neuronal M‐type channels.

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single Km+-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.

Alternative splicing of KCNQ2 potassium channel transcripts contributes to the functional diversity of M-currents

1 The region of alternative splicing in the KCNQ2 potassium channel gene was determined by RNase protection analysis of KCNQ2 mRNA transcripts. 2 Systematic analysis of KCNQ2 alternative splice variant expression in rat superior cervical ganglia revealed multiple variant isoforms. 3 One class of KCNQ2 splice variants, those that contained exon 15a, was found to have significantly different kinetics to those of the other isoforms. These transcripts encoded channel subunits that, when co‐expressed with the KCNQ3 subunit, activated and deactivated approximately 2.5 times more slowly than other isoforms. Deletion of exon 15a in these isoforms produced a reversion to the faster kinetics. 4 Comparison of the kinetic properties of the cloned channel splice variants with those of the native M‐current suggests that alternative splicing of the KCNQ2 gene may contribute to the variation in M‐current kinetics seen in vivo.

ACTIVATION OF NUCLEOTIDE RECEPTORS INHIBITS M-TYPE K CURRENT IK(M) IN NEUROBLASTOMA GLIOMA HYBRID CELLS

A phospholipase-C-linked nucleotide receptor, sensitive to both uridine and adenosine triphosphate (UTP and ATP) has been cloned from NG108-15 neuroblastoma × glioma hybrid cells. We have tested whether activation of this receptor could inhibit the voltage-dependent K+ current [IK(M) or “M-current”] in NG108-15 cells recorded using whole-cell patch-clamp methods. Both UTP and ATP inhibited IK(M) by 44% and 42%, respectively, at 100 μM. Mean IC50 values were: UTP, 0.77±0.27 μM; ATP, 1.81±0.82 μM. The order of nucleotide and nucleoside activity at 100 μM was: UTP = ATP > ATP[γS] = ITP > 2 MeSATP > ADP = GTP ≫ AMP-CPP, adenosine, where ATP[γS] is adenosine 5′-O-(3-thiotriphosphate), ITP is inosine 5′-triphosphate, 2-MeSATP is 2-methylthio ATP and AMP-CPP is α,β methylene ATP. This rank order accords with their activities at the cloned P2U receptor. Effects were not inhibited by suramin (up to 500 μM) or by pre-incubation for 12 h in 500 ng·ml−1Pertussis toxin. Inhibition of IK(M) was frequently preceded by a transient outward current, probably a Ca2+-activated K+ current, responding to Ca2+ mobilization. No effect on the delayed rectifier K+ current was observed. These observations match those expected from stimulating other phospholipase-C-linked receptors in NG108-15 cells.

Ca2+‐inhibited non‐inactivating K+ channels in cultured rat hippocampal pyramidal neurones

1 Whole‐cell perforated‐patch recording from cultured CA1‐CA3 pyramidal neurones from neonatal rat hippocampus (20‐22 °C; [K+]o= 2.5 mM) revealed two previously recorded non‐inactivating (sustained) K+ outward currents: a voltage‐independent ‘leak’ current (Ileak) operating at all negative potentials, and, at potentials ≥−60 mV, a time‐ and voltage‐dependent ‘M‐current’ (IK(M)). Both were inhibited by 1 mM Ba2+ or 10 μM oxotremorine‐M (Oxo‐M). In ruptured‐patch recording using Ca2+‐free pipette solution, Ileak was strongly enhanced, and was inhibited by 1 mM Ba2+ but unaffected by 0.5 mM 4‐aminopyridine (4‐AP), 1 mM tetraethylammonium (TEA) or 1‐10 nM margatoxin. 2 Single channels underlying these currents were sought in cell‐attached patch recordings. A single class of channels of conductance ≈7 pS showing sustained activity at resting potential and above was identified. These normally had a very low open probability (Po < 0.1), which, however, showed a dramatic and reversible increase (to about 0.9 at ≈0 mV) following the removal of Ca2+ from the bath. Under these (Ca2+‐free) conditions, single‐channel Po showed both voltage‐dependent and voltage‐independent components on patch depolarization from resting potential. The mean activation curve was fitted by a modified Boltzmann equation. When tested, all channels were reversibly inhibited by addition of 10 μM Oxo‐M to the bath solution. 3 The channels maintained their high Po in patches excised in inside‐out mode into a Ca2+‐free internal solution and were strongly inhibited by application of Ca2+ to the inner face of the membrane (IC50= 122 nM); this inhibition was observed in the absence of MgATP, and therefore was direct and unrelated to channel phosphorylation/dephosphorylation. 4 Channels in patches excised in outside‐out mode were blocked by 1 mM Ba2+ but were unaffected by 4‐AP or TEA. 5 Channels in cell‐attached patches were inhibited after single spikes, yielding inward ensemble currents lasting several hundred milliseconds. This was prevented in Ca2+‐free solution, implying that it was due to Ca2+ entry. 6 The properties of these channels (block by internal Ca2+ and external Oxo‐M and Ba2+, and the presence of both voltage‐dependent and voltage‐independent components in their Po/V relationship) show points of resemblance to those expected for channels associated with both Ileak and IK(M) components of the sustained macroscopic currents. For this reason we designate them Ksust (‘sustained current’) channels. Inhibition of these channels by Ca2+ entry during an action potential may account for some forms of Ca2+‐dependent after‐depolarization. Their high sensitivity to internal Ca2+ may provide a new, positive feedback mechanism for cell excitation operating at low (near‐resting) [Ca2+]i.

Regulation of M-type Potassium Channels in Mammalian Sympathetic Neurons: Action of Intracellular Calcium on Single Channel Currents

Currents through single M-type potassium channels were recorded in membrane patches excised from rat superior cervical sympathetic neurons. Application of Ca+ to the internal face of inside-out patches produce two forms of M-channel inhibition: a slow, all-or-nothing suppression of activity; and a fast block associated with a concentration-dependent shortening of open times compatible with open-channel block. Both forms of block were enhanced by patch depolarization. Neither was replicated or affected by Mg2+, and both could be recorded in the absence of intracellular ATP, implying that they did not involve phosphorylation. Since the block was reversible in the absence of ATP and since alkaline phosphatase did not reduce channel activity, block was unlikely to have resulted from dephosphorylation. In cell-attached patch recording, M-channel activity increased during exposure of the cell to Ca2(+)-free solution and was rapidly reduced on applying 2mM Ca2+ to the extra-patch solution. This suggests that M-channel activity in these neurons may be tonically regulated by variations in resting intracellular [Ca2+].

Effects of membrane potential and muscarine on potassium M-channel kinetics in rat sympathetic neurones

1. Using cell‐attached patch pipettes, sustained activity of single potassium M‐channels was recorded from dissociated rat superior cervical ganglion neurones. Previous results indicated that this activity, consisting of three main levels of open‐channel conductance (congruent to 7, congruent to 12 and congruent to 19 pS) was activated by membrane depolarization and inhibited by muscarine added outside the patch. Consequently, a kinetic analysis was undertaken in order to identify M‐channel states sensitive to muscarine and membrane potential. 2. Channel activity recorded at 30 mV positive to the resting membrane potential level (congruent to ‐60 mV) showed three shut and two open times. Mean shut times were: tau s1 = 8.0 +/‐ 2.2 ms; tau s2 = 71.3 +/‐ 8.6 ms and tau s3 = 740 +/‐ 220 ms. Mean open times were: tau o1 = 10.6 +/‐ 1.9 ms and tau o2 = 59.3 +/‐ 8.7 ms. When bursts of channel openings were determined as those including tau s1, two exponential components were evident in burst duration distributions (tau b1 = 11.0 +/‐ 0.9 ms and tau b2 = 80.4 +/‐ 11.0 ms). 3. Membrane hyperpolarization significantly lengthened all three shut times and shortened both open times. It also slightly enhanced the relative contribution of high‐conductance channels and decreased the relative contribution of low‐conductance channels to overall activity. 4. All three shut times of the M‐channels were lengthened by 10 microM muscarine without significantly affecting their open times. 5. It is concluded that both open and shut states of the M‐channel are voltage sensitive while only shut states are sensitive to muscarine.