slowpoke
Six mutants of SLO-1, a large-conductance, Ca(2+)-activated K(+) channel of C. elegans, were obtained in a genetic screen for regulators of neurotransmitter release. Mutants were isolated by their ability to suppress lethargy of an unc-64 syntaxin mutant that restricts neurotransmitter release. Evoked postsynaptic currents were measured at the neuromuscular junction in both wild-type and mutants; the removal of SLO-1 greatly increases quantal content primarily by increasing duration of release. The selective isolation of slo-1 as the only ion channel mutant derived from a whole genomic screen to detect regulators of neurotransmitter release suggests that SLO-1 plays an important, if not unique, role in regulating neurotransmitter release (Wang, 2001).
The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which
presynaptic ionic currents and postsynaptic responses can be monitored directly. Cultured embryonic Xenopus
neuromuscular junctions and simultaneous pre- and postsynaptic patch-clamp current-recording procedures were used to identify the major
presynaptic conductances underlying the initiation of neurotransmitter release. Step depolarizations and action potential waveforms
elicit Na and K currents along with Ca2+ and Ca2+-activated K+ (KCa) currents. The onset of KCa current precede the peak of the
action potential. The predominantly omega-CgTX GVIA-sensitive Ca current occurs primarily during the falling phase, but there
is also significant Ca2+ entry during the rising phase of the action potential. The postsynaptic current begins a mean of 0.7 msec
after the time of maximum rate of rise of the Ca2+ current. omega-CgTX also blocks KCa currents and transmitter release during an
action potential, suggesting that Ca and KCa channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp
experiments, KCa channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement
with increasing interpulse interval may reflect the time course of either the deactivation of KCa channels or the diffusion/removal of
Ca2+ from sites of neurotransmitter release after an action potential. It is concluded that the currents carried by the N-type Ca2+ channels and K+ activated Ca2+ channels are functionally coactivated in presynaptic varicosities and coupled to transmitter release (Yazejian, 1997).
The kinetic and steady-state properties of macroscopic mslo (Slowpoke type channel) Ca-activated K+ currents were studied in excised patches from
Xenopus oocytes. In response to voltage steps, the timecourse of both activation and deactivation, but for a brief delay in
activation, can be approximated by a single exponential function over a wide range of voltages and internal Ca2+ concentrations
([Ca]i). Activation rates increase with voltage and with [Ca]i, and approach saturation at high [Ca]i. Deactivation rates generally
decrease with [Ca]i and voltage, and approached saturation at high [Ca]i. Plots of the macroscopic conductance as a function of
voltage (G-V) and the time constant of activation and deactivation shift leftward along the voltage axis with increasing [Ca]i. G-V
relations could be approximated by a Boltzmann function with an equivalent gating charge which ranged between 1.1 and 1.8 e as
[Ca]i varys between 0.84 and 1,000 microM. Hill analysis indicates that at least three Ca2+ binding sites can contribute to channel
activation. Three lines of evidence indicate that there is at least one voltage-dependent unimolecular conformational change
associated with mslo gating that is separate from Ca2+ binding. (a) The position of the mslo G-V relation does not vary
logarithmically with [Ca]i. (b) The macroscopic rate constant of activation approaches saturation at high [Ca]i but remains voltage
dependent. (c) With strong depolarizations mslo currents can be nearly maximally activated without binding Ca2+. These results
can be understood in terms of a channel which must undergo a central voltage-dependent rate limiting conformational change in
order to move from closed to open, with rapid Ca2+ binding to both open and closed states modulating this central step (Cui, 1997).
Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels.
In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage
sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor
pore opening. The presence of voltage sensor rearrangements is demonstrated whose movement and
associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other
voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per
channel is 4-5 elementary charges (Stefani, 1997).
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. Channel activity was studied in cell-attached patches from
hippocampal neurons and a unique specificity of coupling is reported. 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 (Marrion, 1998).
As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca2+ channels and
ryanodine receptors (RyR) in cerebellar granule cells, an examination was carried out to see if such a
coupling could activate a Ca2+-sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar
granule cells. BK channels are specifically activation by group I mGluRs. Group I mGluRs
stimulation of the basal BK channel activity is mimicked by caffeine and both effects were blocked
by ryanodine and nifedipine. Interestingly, carbachol stimulates BK channel activity but through a
pertussis toxin (PTX)-sensitive pathway that is independent of L-type Ca2+ channel activity. This
report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by
mobilizing an additional pathway involving RyR and L-type Ca2+ channels (Chavis, 1998).
The role of individual charged residues of the S4 region of a MaxiK channel (hSlo) in channel gating was investigated. Macroscopic currents induced by wild type (WT) and point mutants of hSlo were investigated in inside-out membrane patches of Xenopus laevis oocytes. Of all the residues tested, only neutralizations of Arg-210 and Arg-213 were associated with a reduction in the number of gating charges as determined using the limiting slope method. Channel activation in WT and mutant channels was interpreted using an allosteric model. Mutations R207Q, R207E, and R210N facilitate channel opening in the absence of Ca2+; however, this facilitation is not observed in the channels Ca2+-bound state. Mutation R213Q behaves similarly to the WT channel in the absence of Ca2+, but Ca2+ is unable to stabilize the open state to the same extent as it does in the WT. Mutations R207Q, R207E, R210N, and R213Q reduce the coupling between Ca2+ binding and channel opening when compared with the WT. Mutations L204R, L204H, Q216R, E219Q, and E219K in the S4 domain show a similar phenotype to the WT channel. It is concluded that the S4 region in the hSlo channel is part of the voltage sensor and that only two charged amino acid residues in this region (Arg-210 and Arg-213) contribute to the gating valence of the channel (Diaz, 1998).
Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I causes the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods reveals two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggests DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increases with DHS-I concentration, and distributions of these mode durations contain two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values are high order with average Hill slopes of 2.4-2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appear to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I causes a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifts the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of -105 mV. 100 nM DHS-I has a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10-20-fold higher affinity than to closed conformations, explains changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels (Giagiacomo, 1998).
BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+
channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to
action potential repolarization in rat adrenal chromaffin cells. In this study, the Ca2+ currents
expressed in these cells are characterized and the Ca2+ channel subtypes are identified that gate the activation of BK
channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four
types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-,
and Q-type Ca2+ currents were 33%, 21%, 12%, and 24% of the total Ca2+ current, respectively.
Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx
through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5
muM nifedipine or Q-type Ca2+ channels by 2 muM Aga IVA reduces BK current activation by 77%
and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2
muM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduces BK current activation
by only 11% and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminate
activation of BK current during action potentials, whereas almost no effects are seen by the selective
blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644
promotes activation of BK current by brief Ca2+ influx steps by more than twofold. These data show
that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel
activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+
channels (Prakriya, 1999).
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 reveal an
inactivation process dependent upon the presence of at least 1 microM intracellular Ca2+. Inactivation
is 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)
consists of very short openings [time constant (tau), approximately 0.14 ms] and rare longer duration
openings (tau, approximately 3.0 ms). Channel inactivation occurs 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 persists whilst recording conditions are constant. Inactivation
is reversed by membrane hyperpolarization, the rate of recovery increasing with further
hyperpolarization and higher extracellular K+. Inactivation is also reversed when the intracellular
Ca2+ concentration is lowered to 100 nM and inactivation is permanently removed by application of trypsin to
the inner patch surface. In addition, inactivation is perturbed by application of either
tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. During
inactivation, channel Po is greater at hyperpolarized rather than depolarized potentials, which is
partly the result of a greater number of longer duration openings. Depolarizing voltage steps (-40 to +40
mV) applied during longer duration openings produces only short duration events at the depolarized
potential, yielding a transient ensemble average current with a rapid decay (tau, approximately 3.8 ms).
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
is removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking
particle may act at the intracellular side of the channel (Hicks, 1998).
Potassium channels play important roles in a wide variety of physiological processes. Although several genes encoding voltage-activated
potassium channels have been analyzed at the molecular level, no calcium-activated potassium channel gene has yet been characterized in
humans. In an effort to provide the foundation for functional analysis of such polypeptides, the cloning of mouse and human
homologs of the Drosophila calcium-activated potassium channel gene, slowpoke, is reported. Both the human and mouse genes
encode polypeptides that have more than 50% amino acid identifies with their Drosophila counterpart. In addition, like the Drosophila
slowpoke gene, both the mouse and human genes generate multiple transcripts by alternative splicing. The human gene maps to
chromosome 10 based on the results of polymerase chain reaction analysis of genomic DNA from human-hamster hybrid cell lines.
Because calcium-activated potassium channels participate in wide variety of cellular functions including neuromuscular communication,
secretion and cellular immunity, their continued analysis promises to have broad biological and medical significance (Pallanck, 1994).
Slo3 is a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homolog) is similar to Slo1, the large conductance, calcium- and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homolog is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testis-specific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility (Schreiber, 1998).
Na+-activated potassium channels (KNa) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. KNa is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native KNa including activation by intracellular Na+, high conductance, and prominent subconductance states. In addition to activation by Na+, rSLO-2 channels are cooperatively activated by intracellular Cl-, similar to C. elegans SLO-2 channels. Since intracellular Na+ and Cl- both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis reveals that the SLO-2 current is a major component of the delayed rectifier. In C. elegans slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily (Yuan, 2003).
Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian
brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila slowpoke gene. By homology screening
with mouse Sla cDNA, a full-length clone (cSlo1) has been isolated from a chick's cochlear cDNA library, rSlol has greater than 90%
identity with mouse Slo at the amino acid level, and is even better matched to a human brain Slo at the amino and carboxy termini.
cSlol has none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain
reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection
of HIEK 293 cells demonstrates that cSlol encodes a potassium channel whose conductance averages 224 pS at +60 mV in symmetrical
140 mM K+. Macroscopic currents through cSlol channels are blocked by scorpion toxin or tetraethyl ammonium, and are voltage and
calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells (Jiang, 1997).
Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of
sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels
involved has been precluded by the small size of the sperm, especially in mammalian species. In the
present study sperm ion channels were expressed in Xenopus oocytes by injection of RNAs of
spermatogenic cells isolated from the rat testes. The RNA-injected oocytes respond to ATP, a
factor known to regulate sperm activation, with the activation of an outwardly rectifying whole-cell
current which was dependent on K+ concentrations and inhibitable by K+ channel blockers,
charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP-induced current can be mimicked
by a Ca2+ ionophore but suppressed by a Ca2+ chelator applied intracellularly, indicating a Ca2+
dependence of the current. Single-channel measurements on RNA-injected oocytes reveals channels
of large conductance which can be blocked by CTX and TEA. Co-injection of germ cell RNAs with
the antisense RNA for a mouse gene encoding slowpoke 'Maxi' Ca2+-activated K+ channels results
in significant reduction of the ATP- and ionomycin-induced current. The expression of the 'Maxi'
Ca2+-activated K+ channels in sperm collected from the rat epididymis was also confirmed by
Western blot analysis. These results suggest that sperm possess Ca2+-activated K+ channels which
may be involved in the process of sperm activation (Chan, 1998).
The Slack gene encodes a voltage-dependent K(+) channel that has a unitary conductance of approximately 60 pS. Evidence from heterologous expression studies suggests that Slack channel subunits can also combine with the Slo subunit to generate Ca(2+)-activated K(+) channels of larger conductances. Nonetheless, the function of Slack in the brain remains to be identified. An affinity-purified antibody was generated against the N-terminal of rat Slack, for biochemical and immunohistochemical studies. The antibody recognizes Slack in transiently transfected CHO cells both by immunocytochemistry and by Western blot analysis. The antibody also detects a single band in rat brain membranes. The localization of Slack in rat brain slices was then determined using the antibody. Most prominent Slack immunoreactivity occurs in the brainstem, in particular the trigeminal system and reticular formation, where very intense staining was found in both cell bodies and axonal fibers of associated nuclei. Labeling was also very strong in the vestibular and oculomotor nuclei. Within the auditory system, the medial nucleus of the trapezoid had a robust signal consistent with staining of the giant presynaptic terminals. Strong Slack immunoreactivity is present in the olfactory bulb, red nucleus, and deep cerebellar nuclei. There was labeling also in the thalamus, substantia nigra, and amygdala. The only cortical region in which Slack immunoreactivity is detected is the frontal cortex. The subcellular and regional distribution of Slack differs from that previously reported for the Slo channel subunit and suggests that Slack may also have an autonomous role in regulating the firing properties of neurons (Bhattacharjee, 2002).
Nine Ca2+-activated K+ channel isoforms from human brain have been cloned and expressed. The open reading frames encode proteins
ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse.
All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 termed hslo. RNA splicing occurs at
four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo
mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in
Xenopus oocytes produces robust voltage and Ca2+-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity,
suggesting a broad functional role for these channels in the regulation of neuronal excitability (Tseng-Crank, 1994).
A family of alternatively spliced cDNAs has been cloned from the receptor epithelium of the chick cochlea. The cDNAs encode a Ca2+-activated K+ channel like those shown to help determine the resonant frequency of electrically tuned hair cells. PCRs using template RNAs from both tonotopically subdivide receptor epithelia and single hair cells demonstrate
differential exon usage along the frequency axis of the epithelium at multiple splice sites of chicken Slowpoke type channels. Single hair cells
express more than one splice variant at a given splice site. Since channel isoforms encoded by differentially spliced slo transcripts
in other species are functionally heterogeneous, these data suggest that differential processing of slo transcripts may account, at
least in part, for the systematic variation in hair-cell membrane properties along the frequency axis of electrically tuned auditory receptor epithelia (Navaratnam, 1997).
Cochlear frequency selectivity in lower vertebrates arises in part from electrical tuning intrinsic to the
sensory hair cells. The resonant frequency is determined largely by the gating kinetics of
calcium-activated potassium (BK) channels encoded by the slo gene. Alternative splicing of slo from
chick cochlea generated kinetically distinct BK channels. Combination with accessory beta subunits
slows the gating kinetics of alpha splice variants but preserves relative differences between them. In
situ hybridization shows that the beta subunit is preferentially expressed by low-frequency (apical)
hair cells in the avian cochlea. Interaction of beta with alpha splice variants could provide the kinetic
range needed for electrical tuning of cochlear hair cells (Ramanathan, 1999).
The effect of ATP in the regulation of two closely related cloned mouse brain large
conductance calcium- and voltage-activated potassium (BK) channel alpha-subunit variants, expressed
in human embryonic kidney (HEK 293) cells, was investigated using the excised inside-out configuration of the
patch-clamp technique. The mB2 BK channel alpha-subunit variant expressed alone is potently
inhibited by application of ATP to the intracellular surface of the patch with an IC50 of 30 muM. The
effect of ATP was largely independent of protein phosphorylation events as the effect of ATP is
mimicked by the non-hydrolysable analogue 5'-adenylylimidodiphosphate (AMP-PNP) and the
inhibitory effect of ATPgammaS is reversible. In contrast, under identical conditions, direct
nucleotide inhibition is not observed in the closely related mouse brain BK channel alpha-subunit
variant mbr5. Furthermore, direct nucleotide regulation is not observed when mB2 is functionally
coupled to regulatory beta-subunits. These data suggest that the mB2 alpha-subunit splice variant
could provide a dynamic link between cellular metabolism and cell excitability (Clark, 1999).
slowpoke Evolutionary homologs part 2/2
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.