Unzipping ion channels.

The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.

Alternative splicing switches potassium channel sensitivity to protein phosphorylation.

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.

A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels.

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Simultaneous binding of two protein kinases to a calcium-dependent potassium channel.

Large-conductance calcium-dependent potassium channels are subject to modulation by protein kinases, phosphatases, and other signaling proteins, and it has been inferred from electrophysiological experiments that signaling proteins sometimes can be intimately associated with these channels in a regulatory complex. We show here that endogenous protein kinase activity coimmunoprecipitates with both native and recombinant Drosophila Slowpoke (dSlo) calcium-dependent potassium channels. Coimmunoprecipitation experiments using antibodies against several protein kinases demonstrate that dSlo can bind simultaneously to the Src tyrosine kinase and to the catalytic subunit of the cAMP-dependent protein kinase (PKAc). Both kinases can phosphorylate the channel in Drosophila heads and in heterologous host cells. The PKAc binds directly to a 172-amino acid region in the C-terminal domain of dSlo, without the intervention of regulatory subunits or anchoring proteins, and channel phosphorylation by PKAc is not required for this binding interaction. In contrast, several phosphorylatable tyrosine residues in dSlo are important for Src binding. The results are consistent with the idea that an ion channel can act as a scaffold for its own specific set of modulatory enzymes.

A dynamically regulated 14-3-3, Slob, and Slowpoke potassium channel complex in Drosophila presynaptic nerve terminals.

Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

Modulation of olfactory bulb neuron potassium current by tyrosine phosphorylation.

Insulin causes a suppression of whole-cell voltage-dependent outward current in cultured neurons from the rat olfactory bulb. This suppression is time-dependent; it is mimicked by application of Src tyrosine kinase inside the cell via the whole-cell patch electrode or by treatment of the olfactory bulb neurons with the tyrosine phosphatase inhibitor pervanadate. The C-type inactivation properties of the outward current in olfactory bulb neurons resemble those of the cloned Kv1.3 potassium channel. In addition, at picomolar concentrations at which it is specific for Kv1.3, the scorpion toxin margatoxin blocks most of the olfactory bulb neuron outward current. Immunocytochemical analysis demonstrates that Kv1.3 is prominent in the cultured olfactory bulb neurons. To identify specific amino acid residues that might be important for potassium current modulation, we examined the effects of pervanadate and insulin on wild-type and mutant Kv1.3 channels expressed in human embryonic kidney (HEK 293) cells. As shown previously, treatment with either pervanadate or insulin suppresses Kv1.3 current in these cells. Mutational analysis demonstrates that at least two distinct tyrosine residues are required for current suppression by pervanadate. Insulin treatment stimulates the tyrosine phosphorylation of Kv1.3 in HEK 293 cells, and a different combination of tyrosine residues is required for the current suppression by insulin. The results suggest that complex patterns of phosphorylation may be involved in the modulation of neuronal potassium current by receptor and nonreceptor tyrosine kinases.

Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel.

Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins.

Expression of voltage-gated potassium channels decreases cellular protein tyrosine phosphorylation.

Protein tyrosine phosphorylation by endogenous and expressed tyrosine kinases is reduced markedly by the expression of functional voltage-gated potassium (Kv) channels. The levels of tyrosine kinase protein and cellular protein substrates are unaffected, consistent with a reduction in tyrosine phosphorylation that results from inhibition of protein tyrosine kinase activity. The attenuation of protein tyrosine phosphorylation is correlated with the gating properties of expressed wild-type and mutant Kv channels. Furthermore, cellular protein tyrosine phosphorylation is reduced within minutes by acute treatment with the electrogenic potassium ionophore valinomycin. Because tyrosine phosphorylation in turn influences Kv channel activity, these results suggest that reciprocal modulatory interactions occur between Kv channel and protein tyrosine phosphorylation signaling pathways.

Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases.

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (alpha-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the alpha-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel.

The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src-induced suppression of current is accompanied by a robust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src-induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src-induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.

Characterization of gating and peptide block of mSlo, a cloned calcium-dependent potassium channel.

The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A "triple mutant" mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism.

Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.

The human Kv1.5 potassium channel (hKv1.5) contains proline-rich sequences identical to those that bind to Src homology 3 (SH3) domains. Direct association of the Src tyrosine kinase with cloned hKv1.5 and native hKv1.5 in human myocardium was observed. This interaction was mediated by the proline-rich motif of hKv1.5 and the SH3 domain of Src. Furthermore, hKv1.5 was tyrosine phosphorylated, and the channel current was suppressed, in cells coexpressing v-Src. These results provide direct biochemical evidence for a signaling complex composed of a potassium channel and a protein tyrosine kinase.

Tyrosine phosphorylation of the Kv1.3 potassium channel.

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.

Ag, a novel protein secreted from Aplysia glia.

We have isolated a cDNA (ag for Aplysia glial) corresponding to an mRNA specific to the nervous system of Aplysia californica. In this study, we characterized the ag cDNA sequence and the distribution of ag mRNA and protein in the Aplysia nervous system. The ag cDNA contains an open reading frame that encodes a novel 29 kD protein. In situ hybridizations demonstrate that ag mRNA is conspicuously absent from the cell bodies of the large neurons constituting the external layer of the ganglia. Instead, it is largely confined to a subset of small, apparently non-neuronal cells surrounding the neurons at the border of the neuropil, is sparsely scattered within the neuropil, and is widespread within the connective nerves, a pattern consistent with glial localization. Polyclonal anti-ag antiserum recognizes a protein between 27 and 29 kD that is more broadly distributed, especially within the neuropil. The distributions of ag mRNA and protein, together with the presence of a putative signal peptide, suggest that ag protein is secreted. Two findings support this hypothesis: first, ag protein is detectable by western blot in Aplysia hemolymph. Second, full length ag protein expressed in COS cells is secreted, but ag lacking the putative signal peptide is not. Secretion from glia raises the possibility that this abundant protein may affect neighboring neurons.

Kinetic variability and modulation of dSlo, a cloned calcium-dependent potassium channel.

We have examined, using patch recording, the modulation by ATP gamma S of the cloned Drosophila slopoke calcium-dependent potassium channel (dSlo) expressed in Xenopus oocytes. There is a large variation in the gating kinetics, open probability, and conductance level of the channel in this expression system, which complicates the analysis of modulatory events. Addition of ATP gamma S to the intracellular face of the patch does not consistently alter the overall open probability of dSlo, but it does increase the frequency of appearance of an exceptionally long-lived closed state of the channel. This modulation is not blocked by an inhibitor of several serine/threonine protein kinases, nor by mutation of a serine residue that is a target for phosphorylation by protein kinase A. Thus, ATP gamma S may alter dSlo kinetic properties by some mechanism other than serine/threonine phosphorylation.

State-dependent modulation of mSlo, a cloned calcium-dependent potassium channel.

The mouse slopoke calcium-dependent potassium channel (mSlo) has been expressed heterologously in COS cells, and incorporated from COS cell membranes into artificial phospholipid bilayers. Under control conditions, the channel is not modulated by ATP. However, when mSlo is treated first with the calcium-dependent potassium channel opener NS004, which itself increases the open probability of the channel, subsequent addition of ATP causes a large further increase in channel open probability. An increase in channel activity is not by itself sufficient to confer sensitivity to ATP, because ATP does not modulate channels whose open probability has been increased by elevated calcium or depolarized voltage. The ATP analog AMP-PNP has only minimal effects on channel activity after treatment with NS004, suggesting that hydrolysis of the ATP is required for its action on mSlo. A peptide inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII) blocks the modulation of mSlo by ATP, whereas peptide inhibitors of other serine/threonine protein kinases are without effect. The results are consistent with a state-dependent modulation of mSlo by ATP, possibly via phosphorylation.