A human intermediate conductance calcium-activated potassium channel

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel

N-type Ca2+ channels can be inhibited by neurotransmitter-induced release of G protein βγ subunits. Two isoforms of Cav2.2 α1 subunits of N-type calcium channels from rat brain (Cav2.2a and Cav2.2b; initially termed rbB-I and rbB-II) have different functional properties. Unmodulated Cav2.2b channels are in an easily activated “willing” (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins shifts Cav2.2b channels to a difficult to activate “reluctant” (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Cav2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Cav2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Cav2.2b to E as in Cav2.2a converts Cav2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Cav2.2a, E177G, converts it to the W state and restores G protein modulation. We propose that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Cav2.2a by a voltage sensor-trapping mechanism. G protein βγ subunits may produce reluctant channels by a similar molecular mechanism.

Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.

Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism.

Ca(2+)-sensitive kinases are thought to play a role in long-term potentiation (LTP). To test the involvement of Ca2+/calmodulin-dependent kinase II (CaM-K II), truncated, constitutively active form of this kinase was directly injected into CA1 hippocampal pyramidal cells. Inclusion of CaM-K II in the recording pipette resulted in a gradual increase in the size of excitatory postsynaptic currents (EPSCs). No change in evoked responses occurred when the pipette contained heat-inactivated kinase. The effects of CaM-K II mimicked several features of LTP in that it caused a decreased incidence of synaptic failures, an increase in the size of spontaneous EPSCs, and an increase in the amplitude of responses to iontophoretically applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. To determine whether the CaM-K II-induced enhancement and LTP share a common mechanism, occlusion experiments were carried out. The enhancing action of CaM-K II was greatly diminished by prior induction of LTP. In addition, following the increase in synaptic strength by CaM-K II, tetanic stimulation failed to evoke LTP. These findings indicate that CaM-K II alone is sufficient to augment synaptic strength and that this enhancement shares the same underlying mechanism as the enhancement observed with LTP.

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.

Role of essential light chain EF hand domains in calcium binding and regulation of scallop myosin.

The specific Ca2+ binding site that triggers contraction of molluscan muscle requires the presence of an essential light chain (ELC) from a Ca2+ binding myosin. Of the four EF hand-like domains in molluscan ELCs, only domain III has an amino acid sequence predicted to be capable of binding Ca2+. In this report, we have used mutant ELCs to locate the Ca2+ binding site in scallop myosin and to probe the role of the ELC in regulation. Point mutations in domain III of scallop ELC have no effect on Ca2+ binding. Interestingly, scallop and rat cardiac ELC chimeras support Ca2+ binding only if domain I is scallop. These results are nevertheless in agreement with structural studies on a proteolytic fragment of scallop myosin, the regulatory domain. Furthermore, Ca2+ sensitivity of the scallop myosin ATPase requires scallop ELC domain I: ELCs containing cardiac domain I convert scallop myosin to an unregulated molecule whose activity is no longer repressed in the absence of Ca2+. Despite its unusual EF hand domain sequence, our data indicate that the unique and required contribution of molluscan ELCs to Ca2+ binding and regulation of molluscan myosins resides exclusively in domain I.

Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral protease, calpain) in systemic rheumatic diseases.

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.