slowpoke
Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels.
They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four
additional hydrophobic regions (S7-S10) of unknown topology. A new model has been proposed where
MaxiK channels have an additional transmembrane region (S0) at the N-terminus that confers beta subunit regulation. Using transient expression of
epitope tagged MaxiK channels, in vitro translation, functional, and ''in vivo'' reconstitution assays, it is shown that MaxiK
channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in
voltage-gated ion channels. Hydrophobic segments S9-S10 in the C terminus are cytoplasmic and
it is unequivocally demonstrated that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus (Meera, 1997).
The Slowpoke-related high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo
channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo
subunit has two parts: a 'core' resembling a subunit from a voltage-dependent K+ channel, and an appended 'tail' that plays a role
in calcium sensing. Evidence is presented for a site on the tail that interacts with calcium. This site, the 'calcium bowl', is a
novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two
categories: (1) those that shift the position of the G-V relation a similar amount at all Ca2+, and (2) those that shift the position of the
G-V relation only at low Ca2+. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in
calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is
sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is
inferred because channels still respond to increasing levels of Ca2+ or [Cd2+], even when the calcium bowl is mutationally
deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly
because of activation of this second Ca2+-binding site (Schreiber, 1997).
A full length alpha-subunit of the Ca2+-activated K+ (BK) channel with an inactivating mutation in
the C-terminus can complement a functional C-terminal fragment. Deletions and amino
acid changes within the S8-S9 interdomain region were analyzed for their ability to allow complementation. Cys612
and His616 that are located in a region that contains two overlapping signature sequences, a
immunoglobulin signature sequence and a heme binding domain, are essential for a functional channel.
These two amino acid residues are also essential for complementation. The deletion of the PEST
sequence does not affect the function of the BK channel; however, without the PEST sequence,
complementation by a functional C-terminal fragments is no longer possible. The ability to complement
a functional channel is restricted to the C-terminal fragment and requires that the complete
alpha-subunit or the larger N-terminal fragment contains both, the immunoglobulin signature sequence
the PEST sequence (Wood, 1997).
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 (Sullivan, 1997).
The high-conductance Ca2+-activated K+ (maxi-K) channel from bovine tracheal smooth muscle was purified to apparent
homogeneity by a combination of conventional chromatographic techniques and sucrose density gradient centrifugation. Fractions
with the highest specific activity for binding of monoiodotyrosine charybdotoxin, [125I]ChTX, were enriched approximately
2000-fold over the initial digitonin-solubilized material up to a specific activity of 1 nmol/mg protein. Silver staining after
SDS-polyacrylamide gel electrophoresis of the fractions from the last step of the purification indicates that binding activity is
correlated with a major component of the preparation that displays an apparent molecular weight of 62,000. Labeling the same
preparation with 125I-Bolton-Hunter reagent reveals the existence of both 62 (alpha)- and 31 (beta)-kDa subunits, in an apparent
stoichiometry of 1:1, comigrating with binding activity. The beta subunit is heavily glycosylated. Deglycosylation studies indicate
that the beta subunit represents the protein to which [125I]ChTX is covalently incorporated in the presence of the bifunctional
cross-linking reagent disuccinimidyl suberate. Binding of [125I]ChTX to the purified ChTX receptor displayed the same
pharmacological profile that has been found previously for toxin binding to native membranes, including inhibition by iberiotoxin,
limbatustoxin, tetraethylamonium, potassium, cesium, and barium. The purified preparation was reconstituted into liposomes which
were then fused with artificial lipid bilayers. Single channels were readily observed with a conductance of 235 picosiemens in 150
mM KCl that displayed selectivity for potassium over chloride and that were blocked by ChTX. The open probability of these
channels was increased by depolarizing membrane potentials and by raising the internal calcium concentration. These data
suggest that the maxi-K channel purified from tracheal smooth muscle is composed of two subunits (Garcia-Calvo, 1994).
Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel
leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when
the alpha subunit is expressed alone. To
identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala
scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on
125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of
the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting
that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the
extracellular loop of beta was demonstrated after comparison of reactivities of native beta and
single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta,
when mutated individually or in combinations, has no effect on toxin binding with the exception of
Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis
studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90,
Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta
subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association
and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these
data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a
restricted conformation. Moreover, they are consistent with the view that four residues appear to be
important for inducing an appropriate conformation within the alpha subunit that allows high affinity
ChTX binding (Hanner, 1998).
Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance
Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel.
Using single-channel analysis to investigate the mechanism for this increase, it was found that the beta
subunit increases open probability (Po) by increasing burst duration 20-100-fold, while having little
effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected
open and closed states entered during gating. The effect of the beta subunit is not equivalent to
raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not
act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibits transitions to
subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit
that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each
burst of openings is greatly amplified in duration through increases in both the numbers of openings per
burst and in the mean open times. Native BK channels from cultured rat skeletal muscle
have bursting kinetics similar to channels expressed from alpha subunits alone (Nimigean, 1999).
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle and other cell types and are important determinants of smooth muscle tone. To determine the
mechanisms involved in the transcriptional regulation of maxiK channels, the
promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel
complex were characterized. Maximum promoter activity (up to 12.3-fold over control) occurs between nucleotides -567
and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites
and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1,
PEA-3, Myo-D, and E2A, are observed in the 5'-flanking sequence. Additionally, a CCTCCC
sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located
27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking
region. However, the promoter directs equivalent expression when transfected into smooth muscle
and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at
position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap
site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this
sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific
transcriptional activity is not observed. The lack of tissue-specific promoter activity, particularly the
finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a
complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the
promoters of the two genes suggests that regulation of coordinate expression of the subunits does not
occur through equivalent cis-acting sequences (Dhulipala, 1999).
Voltage- and Ca2+-sensitive K+ (MaxiK) channels play key roles in controlling neuronal excitability
and vascular tone. Human and rodent genes for the modulatory beta subunit,
KCNMB1, have been cloned and analyzed. The human and mouse beta-subunit genes are approximately 11 and approximately 9 kb in
length, respectively, and have a four exon-three intron structure. Primer extension assay localized the
transcription initiation site at 442 (human) or 440 (mouse) bp upstream of the translation initiation
codon, agreeing with the transcript size in Northern blots. Both genes have a TATA-less putative
promoter region, with a transcription initiator-like region, and motifs characteristic of regulated
promoters, including muscle-specific enhancing factors-1 and -2. Consistent with a tissue-specific
expression of KCNMB1, regulated at the transcriptional level, beta-subunit transcripts are abundant in
smooth muscle and heart, but scarce in lymphatic tissues, brain, and liver. Expressed rat and mouse
beta subunits increase the apparent Ca2+ sensitivity of the human MaxiK channel alpha subunit (Jiang, 1999).
Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. A transmembrane beta subunit (beta2) is reported that yields inactivating MaxiK currents on coexpression with the pore-forming alpha subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the beta2 subunit abolishes inactivation of the alpha subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle beta1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the beta2 subunit causes inactivation of the MaxiK channel alpha subunit by occluding its K+-conducting pore resembling the inactivation caused by the 'ball' peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different beta subunits (Wallner, 1999).
The human large conductance, calcium-activated potassium (maxi-K) channel (alpha and beta
subunits) and beta2-adrenergic receptor genes were coexpressed in Xenopus oocytes in order to study
the mechanism of beta-adrenergic modulation of channel function. Isoproterenol and forskolin
increase maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and
both channel subunits by 33% +/- 5% and 35% +/- 8%, respectively, without affecting current activation or
inactivation. The percentage of stimulation by isoproterenol and forskolin is not different in oocytes
coexpressing the alpha and beta subunits versus those expressing the only the alpha subunit, suggesting
that the alpha subunit is the target for regulation. The stimulatory effect of isoproterenol is almost
completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK)
regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide has
little effect, indicating that cellular coupling of beta2-adrenergic receptors to maxi-K channels involves
endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation
sites (serine 869) on the alpha subunit almost completely inhibits beta-adrenergic receptor/channel
stimulatory coupling, whereas forskolin still stimulates currents moderately (16% +/- 4%). These data
demonstrate that physiological coupling between beta2 receptors and maxi-K channels occurs by the
cAMP-PK mediated phosphorylation of serine 869 on the alpha subunit on the channel (Nara, 1998).
The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese
hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit
(CHOalpha+beta cells). Basic channel properties and the effects of cGMP- and
cAMP-dependent protein kinases on the BK channel activity were studied. Coexpression of alpha and beta subunits
enhance the Ca2+ and voltage sensitivity of the BK channel, and decrease the inhibitory potency of
iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide,
respectively, are independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase fail
to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different Ca2+i and
voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women
respond to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open
probability (NPo), respectively. These effects can be reversed by alkaline phosphatase and remain
unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however,
cAMP and cGMP kinases increase NPo 2-fold and 3.5-fold, respectively. This stimulation is
enhanced rather than reversed by alkaline phosphatase and is abolished by 100 nM okadaic acid.
The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by
cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory
regulation of BK channels by cAMP kinase and cGMP kinase is observed, suggesting that channel
regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively,
failure of regulation may have been due to the primary structure of the myometrial BK channel protein
used in this study (Zhou, 1998).
Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by
cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1)
direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase.
To scrutinize the first possibility, at the molecular level, the human pore-forming alpha-subunit
of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I were used. In
cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP
significantly increases the macroscopic currents. This increase in current is due to an increase in the
channel voltage sensitivity by approximately 20 mV and is reversed by alkaline phosphatase
treatment after patch excision. In inside-out patches, however, the effect of purified kinase was
negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase
applied to the cytoplasmic side of reconstituted channels increases their open probability. This
stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show
that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately
125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation
experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+
channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for
PKG-Ialpha-induced activation of maxi-K channels (Alioua, 1999).
The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance
calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive
action potentials (spike-frequency adaptation) and is essential for normal neurotransmission. SK channels are
voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium
sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential.
The mechanism of calcium gating has been studied and SK channels are found not to be gated by calcium binding directly to the channel
alpha-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively
associated with the alpha-subunits in a calcium-independent manner. These data support a model in which calcium gating of SK
channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein (Xia, 1998a).
Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in
the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which
are sensitive to both membrane potential and intracellular calcium. A potassium
channel gene called Slack has been isolated has been isolated from the rat that is abundantly expressed in the nervous system. Slack channels rectify
outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium.
However, when Slack is co-expressed with Slo, channels with pharmacological properties and
single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels
have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. These
findings indicate that some intermediate-conductance channels in the nervous system may result from
an interaction between Slack and Slo channel subunits (Joiner, 1998).
slowpoke Evolutionary homologs part 1/2
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.