False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions.

The techniques of co-immunoprecipitation and immunocytochemical co-labelling are classically used to identify protein-protein interactions. We have used an antibody to the rat small conductance calcium-activated potassium channel subtype 1 (rSK1) to immunoprecipitate proteins from rat brain. A 35 kDa protein was recognized by two monoclonal antibodies to syntaxin 1 and a polyclonal antibody to syntaxin 1A, but not by antibodies to syntaxins 2, 3 or 4. These data suggested that syntaxin 1A is specifically associated with rSK1 in rat brain. A GST construct of the carboxyl terminus of rSK1 was able to pull-down syntaxin 1A from rat brain. Immunocytochemistry showed somatic labelling for both rSK1 and syntaxin 1A in acutely dissociated hippocampal CA1 neurons, confirming that these proteins could interact in vivo. However, control immunoprecipitations showed that antibodies to eight potassium channels could also immunoprecipitate syntaxin, even though some of these channels would not be expected to reside in the same subcellular compartment. Mock immunoprecipitations and pull-down assays showed that syntaxin 1 could directly interact with sepharose and agarose resins. Hence immunoprecipitation and pull-down assays do not provide evidence that syntaxin is specifically associating with a protein, placing doubt on a number of reported interactions with syntaxin 1A.

Somatic colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal neurons.

In hippocampal neurons, the firing of a train of action potentials is terminated by generation of the slow afterhyperpolarization (AHP). Recordings from hippocampal slices have shown that the slow AHP likely results from the activation of small-conductance calcium-activated potassium (SK) channels by calcium (Ca(2+)) entry through L-type Ca(2+) channels. However, the relative localization of these two channel subtypes is not known. The cloning and characterization of three subtypes of SK channel has suggested that SK1 may underlie generation of the slow AHP. Using a novel antibody directed against rat SK1 (rSK1), it has been determined that the rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In conjunction with antibodies directed against C (Ca(v)1.2) and D (Ca(v)1.3) class L-type Ca(2+) channel alpha1 subunits, it was observed that rSK1 channels were selectively colocalized with D class L-type channels. This colocalization supports the functional coupling of L-type and SK channels previously observed in cell-attached patches from hippocampal neurons. However, it appears contrary to the slow rise and decay of the slow AHP. Induction of delayed facilitation of L-type Ca(2+) channels in cell-attached patches from hippocampal neurons evoked delayed opening of coupled SK channels. Generation of ensemble currents produced waveforms identical to the ionic current underlying the slow AHP (I(sAHP)). Therefore, these data indicate that the slow AHP is somatic in origin, resulting from delayed facilitation of D class L-type Ca(2+) channels colocalized with rSK1 channels.

Gating properties of single SK channels in hippocampal CA1 pyramidal neurons.

The activation of small-conductance calcium-activated potassium channels (SK) has a profound effect on membrane excitability. In hippocampal pyramidal neurons, SK channel activation by Ca2+ entry from a preceding burst of action potentials generates the slow afterhyperpolarization (AHP). Stimulation of a number of receptor types suppresses the slow AHP, inhibiting spike frequency adaptation and causing these neurons to fire tonically. Little is known of the gating properties of native SK channels in CNS neurons. By using excised inside-out patches, a small-amplitude channel has been resolved that was half-activated by approximately 0.6 microM Ca2+ in a voltage-independent manner. The channel possessed a slope conductance of 10 pS and exhibited nonstationary gating. These properties are in accord with those of cloned SK channels. The measured Ca2+ sensitivity of hippocampal SK channels suggests that the slow AHP is generated by activation of SK channels from a local rise of intracellular Ca2+.

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons.

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.

Calcium-activated potassium channels.

Calcium-activated potassium channels are fundamental regulators of neuronal excitability, participating in interspike interval and spike-frequency adaptation. For large-conductance calcium-activated potassium (BK) channels, recent experiments have illuminated the fundamental biophysical mechanisms of gating, demonstrating that BK channels are voltage gated and calcium modulated. Structurally, BK channels have been shown to possess an extracellular amino-terminal domain, different from other potassium channels. Domains and residues involved in calcium-gating, and perhaps calcium binding itself, have been identified. For small- and intermediate-conductance calcium-activated potassium channels, SK and IK channels, clones have only recently become available, and they show that SK channels are a distinct subfamily of potassium channels. The biophysical properties of SK channels demonstrate that kinetic differences between apamin-sensitive and apamin-insensitive slow afterhyperpolarizations are not attributable to intrinsic gating differences between the two subtypes. Interestingly, SK and IK channels may prove effective drug targets for diseases such as myotonic muscular dystrophy and sickle cell anemia.

Ca2+-dependent inactivation of large conductance Ca2+-activated K+ (BK) channels in rat hippocampal neurones produced by pore block from an associated particle.

1. Recordings of the activity of the large conductance Ca2+-activated K+ (BK) channel from over 90 % of inside-out patches excised from acutely dissociated hippocampal CA1 neurones revealed an inactivation process dependent upon the presence of at least 1 microM intracellular Ca2+. Inactivation was characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consisted of very short openings (time constant (tau), approximately 0.14 ms) and rare longer duration openings (tau, approximately 3.0 ms). 2. Channel inactivation occurred with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside-out recording. Inactivation persisted whilst recording conditions were constant. 3. Inactivation was reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation was also reversed when the intracellular Ca2+ concentration was lowered to 100 nM and was permanently removed by application of trypsin to the inner patch surface. In addition, inactivation was perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. 4. During inactivation, channel Po was greater at hyperpolarized rather than depolarized potentials, which was partly the result of a greater number of longer duration openings. Depolarizing voltage steps (-40 to +40 mV) applied during longer duration openings produced only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (tau, approximately 3.8 ms). 5. These data suggest that hippocampal BK channels exhibit a Ca2+-dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation was removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking particle may act at the intracellular side of the channel.

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.

Gating of recombinant small-conductance Ca-activated K+ channels by calcium.

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 micro M Ca2+ and was maximal above 2 micro M Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was approximately 0.6 in 1 micro M free Ca2+. A lower open probability of approximately 0.05 in 1 micro M Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

Beta-adrenergic stimulation selectively inhibits long-lasting L-type calcium channel facilitation in hippocampal pyramidal neurons.

L-type calcium channels are abundant in hippocampal pyramidal neurons and are highly clustered at the base of the major dendrites. However, little is known of their function in these neurons. Single-channel recording using a low concentration of permeant ion reveals a long-lasting facilitation of L-type channel activity that is induced by a depolarizing prepulse or a train of action potential waveforms. This facilitation exhibits a slow rise, peaking 0.5-1 sec after the train and decaying over several seconds. We have termed this behavior "delayed facilitation," because of the slow onset. Delayed facilitation results from an increase in opening frequency and the recruitment of longer duration openings. This behavior is observed at all membrane potentials between -20 and -60 mV, with the induction and magnitude of facilitation being insensitive to voltage. beta-Adrenergic receptor activation blocks induction of delayed facilitation but does not significantly affect normal L-type channel activity. Delayed facilitation of L-type calcium channels provides a prolonged source of calcium entry at negative membrane potentials. This behavior may underlie calcium-dependent events that are inhibited by beta-adrenergic receptor activation, such as the slow afterhyperpolarization in hippocampal neurons.

Control of M-current.

M-current is a non-inactivating potassium current found in many neuronal cell types. In each cell type, it is dominant in controlling membrane excitability by being the only sustained current in the range of action potential initiation. It can be modulated by a large array of receptor types, and the modulation can occur either by suppression or enhancement. Modulation of M-current has dramatic effects on neuronal excitability. This review discusses the numerous second messenger pathways that converge on regulation of this current: in particular, two forms of regulation of the M-current, receptor-mediated modulation and the control of macroscopic current amplitude by intracellular calcium. Both types of regulation are discussed with reference to the modulation of single-channel gating properties.

Small-conductance, calcium-activated potassium channels from mammalian brain.

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Conduction properties of the M-channel in rat sympathetic neurons.

We have investigated the conduction properties of the M-channel in rat superior cervical ganglion neurons. Reversal potentials measured under bi-ionic conditions yielded a permeation sequence of Tl > K > Rb > Cs > NH4 > Na. Slope conductances gave a conductance sequence of K > Tl > NH4 > Rb > Cs. M-current was shown to exhibit a number of features atypical of potassium channels. First, the conduction of monovalent cations relative to K was very low. Second, the nature of the permeant ion did not affect the deactivation kinetics. Third, M-current did not exhibit anomalous mole-fraction behavior, a property suggestive of a multi-ion pore. Finally, external Ba, which is a blocker of M-current, showed a preferential block of outward current and had much less effect on inward current. The permeability sequence of the M-channel is very similar to other K-selective channels, implying a high degree of conservation in the selectivity filter. However, other conduction properties suggest that the pore structure outside of the selectivity filter is very different from previously cloned potassium channels.

Calcineurin regulates M channel modal gating in sympathetic neurons.

The M current regulates neuronal excitability, with its amplitude resulting from high open probability modal M channel behavior. The M current is affected by changing intracellular calcium levels. It is proposed that internal calcium acts by regulating M channel modal gating. Intracellular application of a preactivated form of the calcium-dependent phosphatase calcineurin (CaN420) inhibited the macroscopic M current, while its application to excised inside-out patches reduced high open probability M channel activity. Addition of ATP reversed the action of CaN420 on excised patches. The change in M channel gating induced by CaN420 was different from the effect of muscarine. A kinetic model supports the proposition that shifts in channel gating induced by calcium-dependent phosphorylation and dephosphorylation control M current amplitude.

Properties of Kv2.1 K+ channels expressed in transfected mammalian cells.

We have transiently expressed the rat Kv2.1 K+ channel polypeptide (pKv2.1) at high levels by transfection of mammalian COS-1 cells. Kv2.1-transfected cells express a molecular mass of 108 kDa pKv2.1, larger than the size of the core polypeptide (95 kDa) predicted from the deduced primary sequence and of pKv2.1 synthesized in cell free or Xenopus oocyte translation systems. The increased size of pKv2.1 in COS-1 cells is due to a posttranslational modification that occurs early (t1/2 = 5 min) in the biosynthetic transport through the endomembrane system, presumably while the protein resides in the endoplasmic reticulum. The increased size is entirely due to phosphorylation, based on in vivo 32P-labeling and sensitivity to alkaline phosphatase digestion. Immunofluorescent localization of pKv2.1 shows intense surface labeling; no intracellular pools of retained protein are apparent. Immunogold electron microscopy confirms that the expressed polypeptide is found on the cell surface in small clusters or patches of 10-15 gold particles. Cells expressing pKv2.1 exhibit large, voltage-dependent outward currents. The pharmacological properties of the expressed Kv2.1 currents are virtually indistinguishable from those described previously in Xenopus oocytes microinjected with Kv2.1 cRNA, but differences in voltage-dependent properties were observed. High level of expression of functional pKv2.1 in these cells points to the utility of this system for the rapid biochemical, cell biological and electrophysiological analysis of altered forms of pKv2.1, and other members of the K+ channel gene family.

M-current suppression by agonist and phorbol ester in bullfrog sympathetic neurons.

Activation of protein kinase C (PKC) by phorbol esters is known to suppress M-current. 4-beta-Phorbol 12,13-dibutyrate (PDBu) irreversibly suppressed M-current in a concentration-dependent manner (Ki 38 nM). Inhibitors of PKC, the pseudo-substrate peptide PKCI (19-31), staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) antagonized PDBu-mediated suppression of M-current. Suppression of M-current by muscarine and luteinizing hormone-releasing hormone (LHRH) was unaffected by PKCI (19-31) and H7, but was antagonized by staurosporine. The balance of data suggests that suppression of M-current by agonists is probably not mediated by activation of PKC. Addition and subsequent removal of PDBu to M-current suppressed by muscarine prevented the action of PDBu, while closing M-channels by voltage or blocking by barium did not. This suggests that M-channel closure by muscarine protects those channels from the effects of PDBu. Partial suppression of M-current by low concentrations of muscarine antagonized the response to PDBu, with the magnitude of suppression equivalent to that seen with PDBu alone. It is suggested that two interconvertable populations of M-channels exist, one that is sensitive to both agonist and PDBu and another that can only be suppressed by agonist.

Selective reduction of one mode of M-channel gating by muscarine in sympathetic neurons.

M-current is widespread in the nervous system. It stabilizes cell excitability, and its suppression by muscarinic receptor activation underlies slow synaptic transmission in sympathetic neurons. Suppression of M-current was one of the first examples of neuromodulation of a potassium current, but the mechanism is not understood. Single-channel recording was used to study this issue. An M-channel with two conductance states, which exhibited appropriate voltage-dependent kinetics with two modes of gating, has been resolved. Mode 1 comprises short open time, low open probability events, and mode 2 openings represent long open time, high open probability behavior. Muscarine decreased M-channel activity by selectively reducing mode 2 M-channel gating through a diffusible second messenger. It is suggested that control of modal gating may be a widespread mechanism for neuromodulation.

Multiple kinetic states underlying macroscopic M-currents in bullfrog sympathetic neurons.

M-current is a time- and voltage-dependent potassium current which is suppressible by muscarinic receptor activation. We have used curve fitting and noise analysis to determine if macroscopic M-currents deviate from a previously predicted simple two-state kinetic scheme. The M-current was best described by three kinetically distinct components: 'fast' (tau 0), 'intermediate' (tau 1) and 'slow' (tau 2) time constants. The 'fast' (tau 0) and 'intermediate' (tau 1) components were identified from the spectra of M-current noise at potentials positive to the cells' resting membrane potential. The 'intermediate' (tau 1) and 'slow' (tau 2) components were seen by curve fitting M-current deactivation currents. The 'intermediate' (tau 1) time constant was voltage dependent (decreasing e-fold in 23 mV), but voltage dependence of the 'fast' (tau 0) and 'slow' (tau 2) components was not obvious. All kinetic components were sensitive to muscarine, with the 'intermediate' (tau 1) and 'slow' (tau 2) being equally so. These data suggest that all components may derive from the same channel population, and that the M-channel may have at least four kinetic states.

Release of intracellular calcium and modulation of membrane currents by caffeine in bull-frog sympathetic neurones.

1. Calcium release and sequestration were studied in whole-cell voltage-clamped bull-frog sympathetic neurones by image analysis of Fura-2 signals. 2. Application of caffeine (10 mM) to cells voltage clamped at -38 mV caused a rapid increase in intracellular calcium concentration ([Ca2+]i) to a mean value of 352 +/- 33 nM, which activated an outward current. In the continued presence of caffeine the rise in [Ca2+]i slowly declined to a sustained plateau of 196 +/- 20 nM (112 nM above control levels), while the outward current rapidly decayed. Peak calcium release was highest at the edge of the cell. 3. The caffeine-evoked intracellular calcium increase was reduced by two inhibitors of calcium-induced calcium release, ryanodine and procaine. The residual non-suppressible increase in [Ca2+]i may indicate that caffeine can release calcium from two pharmacologically distinct intracellular stores. 4. Inhibition of the caffeine-evoked release of calcium by ryanodine was both concentration and 'use dependent' so that the full inhibitory effect was only observed when caffeine was applied for the second time in the presence of ryanodine. In contrast, the action of procaine did not show any 'use dependence' and unlike ryanodine was fully reversible. 5. The outward current was sensitive to blockers of the large conductance calcium-activated potassium current, Ic. Analysis of variance from this current indicated that it arose at least partly from summation of spontaneous miniature outward currents. 6. The magnitude and duration of calcium release by caffeine was dependent on the resting level of intracellular calcium and the caffeine exposure time. This, together with the pharmacology of the release, suggests that caffeine increases intracellular calcium by sensitizing calcium-induced calcium release. 7. The evoked [Ca2+]i increase was enhanced in amplitude by intracellular application of Ruthenium Red. This effect was mimicked by extracellular application of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) but not by internal application of FCCP or other inhibitors of mitochondrial Ca2+ uptake. This suggests that the evoked increase in [Ca2+]i is predominantly buffered by a Ruthenium Red-sensitive sequestration process which is not mitochondrial.

Modulation of M-current by intracellular Ca2+.

IM is a voltage- and time-dependent K+ current that is suppressed by muscarinic receptor activation. IM augmentation following agonist washout was blocked by heavily buffering [Ca2+]i using BAPTA. Although IM is not primarily Ca2+ dependent, small increases in [Ca2+]i by photolysis of the "caged" Ca2+ chelator nitr-5 or by evoking action potentials augmented, while larger increases inhibited, IM. Raising [Ca2+]i for prolonged periods, by nitr-5 photolysis, reduced its sensitivity to agonist, leaving a poorly reversible response. These results suggest that IM can be regulated by physiologically relevant changes in [Ca2+]i, placing IM in a unique position to modulate cell excitability.