From the Departments of Anatomy and Neurobiology and
§ Genetics, Washington University School of Medicine,
St. Louis, Missouri 63110
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ABSTRACT |
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Potassium channels have evolved to play specialized roles in both excitable and inexcitable tissues. Here we describe the cloning and expression of Slo3, 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. Reverse transcription-polymerase chain reaction, Northern analysis, and in situ hybridization show that 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 homologue) 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 homologue 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.
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INTRODUCTION |
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Many ion channels respond to membrane depolarization or intracellular ligands. In the Slo1 channel, originally cloned from Drosophila, these features are combined in a channel that opens in response to both depolarization and intracellular calcium increases (1, 2). Slo1 channels cloned from mouse and human show strong conservation of sequence and functional properties (3-7). One proposed role of the Slo1 channel is to provide negative feedback for the entry of calcium into cells via hyperpolarization-induced closure of voltage-dependent calcium channels. Perhaps because of the versatility of this mechanism, Slo1 channels are expressed in many tissues where voltage-gated calcium channels are present, including brain, skeletal and smooth muscle, auditory hair cells, pancreas, and adrenal gland (8-15). In contrast to other multigene voltage-gated K+ channel families, Slo1 has remained the sole functionally characterized representative of its family (16). Here we describe mSlo3,1 a pH- and voltage-dependent Slo family member prominently expressed in mouse spermatocytes. Despite similarity in sequence of mSlo3 to mSlo1, mSlo3 is insensitive to calcium over a wide concentration range. Thus, the unifying characteristics of the Slo gene family of channels may be a dual sensitivity to membrane voltage and a variety of intracellular factors, such as [H+] for mSlo3.
The uniquely abundant expression of mSlo3 in developing spermatocytes presents further interesting questions. Spermatocytes require proteins tailored to fulfill roles unique to the process of germ cell development and fertilization. Cellular signaling in spermatic cells is tightly regulated to prevent inappropriate activation of the irreversible steps that prepare the sperm to fertilize the oocyte. Many of these steps are triggered and coordinated by changes in membrane potential and intracellular Ca2+ concentration and pH. Because of the central importance of these events in development, many efforts have been made to identify the specific proteins, including ion channels, which regulate spermatic function. In particular, there have been reports of channels present in spermatocytes (19, 20), including voltage-dependent calcium channels (21-24). In addition, a cyclic nucleotide-gated channel has been directly cloned from testis (25). The mSlo3 channel exhibits a unique combination of voltage dependence and sensitivity to pH that may be important for spermatic function.
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MATERIALS AND METHODS |
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Cloning--
By tBlastn (NCBI), an EST (GenBankTM
accession number AA072586) was identified by homology to the C-terminal
"tail" of mSlo1 (17). The EST originated from a mouse promyelocytic
WEHI-3 cell line cDNA library. A 32P-labeled 1,254-bp
PCR product generated from the EST pBluescript plasmid (Genome Systems)
was employed to isolate cDNA clones from a WEHI-3 library
(Stratagene) by hybridization. The oligonucleotides used to generate
the probe were 5-GTGGATGATACCGACATGCTGGAC-3
(sense) and
5
-GAGACCACCTCTCTCCCGTGTCGT-3
(antisense). mSlo3 expression in the
WEHI-3 cell line is apparently anomalous; all isolated cDNAs were
inappropriately spliced or truncated, and PCR analysis of the WEHI-3
cDNA bank using combinations of primers complementary to mSlo3 and
vector sequence indicated that complete cDNAs were not represented.
Subsequent screening of a mouse testis cDNA library (Dr. Graeme
Mardon) yielded cDNAs that extended to the putative initiator
methionine, as shown in Fig. 1. The reading frame was closed upstream
from the initiator methionine. A full-length cDNA was constructed
from two overlapping cDNAs. The presence of full-length transcripts
in testis corresponding to this cDNA was verified by RT-PCR from
total testis RNA. The entire cDNA was sequenced in both directions.
For expression in Xenopus oocytes, a Kozak initiator
sequence (26) was introduced by PCR, and the entire open reading frame
was subcloned into the pOocyte-Xpress vector (17). cRNA was generated
using the mMessage mMachine kit (Ambion); details of cRNA synthesis
were as previously published (17).
RT-PCR--
For each tissue, Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc.) was used on 5.0 µg of
total RNA primed with 25 µM random hexanucleotides
(Boehringer Mannheim) and 200 µM dNTPs at 42 °C for
1 h. 0.1% of each first strand synthesis was assayed by PCR using
1.0 µM oligonucleotide primers, 200 µM dNTPs, and 0.0075 units of KlenTaq, cycling 30 times. Reaction products
were electrophoresed on 1.5 and 3.0% agarose gels using standard
Tris-borate (TBE) buffer and visualized by staining with ethidium
bromide. PCR primer pairs used were 1) mSlo3 (S4 to S5), 5-CTCGAACTCCCTAAAATCTTACAGAT-3
(sense) and
5
-TTCCGTTGAGCCAGGGGTCACCAGAATT-3
(antisense) to generate a 156-bp
product; 2) mSlo3 (S8 to S9), 5
-TCTGCTTTGTGAAGCTAAATCT-3
(sense) and
5
-TTTCAAAGCCTCTTTAGCGGTAA-3
(antisense) to generate a 690-bp product;
3) mSlo3 (S9 to S10), 5
-TTATGCCTGGATCTGCACTCTACATG-3
(sense) and
5
-ATAGTTTCCGTCTACTACCGAAA-3
(antisense) to generate a 221-bp product;
4) human
-actin, 5
-GATGATATCGCCGCGCTCGTCGTCGAC-3
(sense) and
5
-TCGGTCCAGGTCTGCGTCCTACCGTAC-3
(antisense) to generate a 535-bp
product.
Northern Blot Analysis--
Total RNA was isolated from freshly
dissected mouse tissue using Trizol (Life Technologies, Inc.). 20 µg
of total RNA from each tissue was electrophoresed on a 1% agarose
denaturing gel using MOPS-formaldehyde buffer then transferred to
nitrocellulose. The human tissue blot was obtained commercially
(CLONTECH). A PCR product generated using primers
5-CGGAAACGTCATGTACAATCGAAATCCA-3
(sense) and
5
-TTCCGTTGAGCCAGGGGTCACCAGAATT-3
(antisense) was labeled using random
hexanucleotides (Boehringer Mannheim). Both human and mouse blots
were hybridized and washed under standard high stringency conditions
and exposed to x-ray film for 4-16 h. After hybridization with mSlo3
probes, blots were rehybridized with a human
-actin probe to verify
RNA loading.
In Situ Hybridization--
Testes from white mice >30 days old
were dissected, frozen, sectioned immediately with a cryostat,
collected on slides, and stored at 20 °C. A partial mSlo3 cDNA
(approximately 1 kb, corresponding to coding sequence for residues
170-510) was subcloned into pBluescript II KS+
(Stratagene). T3 and T7 RNA polymerase (Stratagene) were used to
synthesize [33P]UTP-labeled antisense and sense probes,
respectively, from linearized plasmid. Slides were hybridized overnight
at 55 °C. After washing, slides were dipped in NTB-2 liquid emulsion
(Eastman Kodak Co.), air-dried, and placed in light-protected boxes at
4 °C for 10 days.
Electrophysiology--
cRNA (40 nl at approximately 1 µg/µl)
was injected into mature Xenopus oocytes; recordings were
made 1-8 days later. For whole cell recording, medium nd96 (96 mM NaCl, 2 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 5 mM
HEPES, pH 7.5) supplemented with 1 mM DIDS (to block
endogenous chloride currents) was employed as a bath solution or
modified as noted in the figure legends. Patches were perfused with
either zero Ca2+-EGTA solutions (160 mM
potassium gluconate, 34 mM KOH, 10 mM HEPES,
and 10 mM EGTA) or Ca2+-containing (184 mM potassium gluconate, 10 mM KOH, 10 mM HEPES, 200 µM hemicalcium gluconate)
solutions. HCl was used to adjust the pH. The pipette solution
contained 0.5 mM potassium gluconate, 0.5 mM
KCl, 1.1 mM KOH, 10 mM HEPES, 159 mM sodium gluconate, and 2 mM hemimagnesium gluconate, pH
7.1. Whole cell recordings were obtained using the two-electrode
voltage clamp TEV-200 amplifier (Dagan). Patch currents were recorded
on either an Axopatch 1B or 200A amplifier (Axon) and digitized at
either 3.4, 5, or 10 kHz. Data acquisition and analysis programs were
CCURRENT and CQUANT (Dr. Keith Baker) or pClamp6 (Axon). Single-channel
conductance was determined from the slope of the unitary current
amplitude versus voltage relation. The tail current
amplitude versus voltage data (see Fig. 4) were fit with the
sum of two independent Goldman-Hodgkin-Katz current equations. Each
equation has the form I = PVmF2/RT × ([S]i [S]o × EXP(
VmF/RT))/1
e
VmF/RT,
where S is either K+ or Na+.
P, the permeability of Na+ or K+,
was allowed to vary freely. [S]i and
[S]o represent ion concentrations inside and
outside the oocyte, respectively; Vm is the membrane
potential; F, R, and T have their usual meanings. Reversal potential versus varying
extracellular cation concentration (see Fig. 4) was fit with the
Goldman-Hodgkin-Katz equation: Ereversal = RT/F × ln([K+]o + P × [Na+]o)/([K+
]i + P × [Na+]i), where
P is the Na+ to K+ permeability
ratio. Fits were performed using Sigmaplot (Jandel). Recordings were
made at room temperature except tail currents, which were recorded at
11 °C using a Peltier device (Cambion).
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RESULTS |
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Cloning and Primary Sequence of mSlo3-- We isolated the mSlo3 cDNA from a testis cDNA library based on its homology to the large conductance calcium-activated (BK) potassium channel, mSlo1 (see "Materials and Methods"). The probe was generated from an expressed sequence tag identified in the GenBankTM data base. The new channel was termed mSlo3 (the "m" prefix denoting mouse derivation, "h" referring to the human homologue, hSlo3). Fig. 1 illustrates that the 1,112 amino acid mSlo3 protein is similar along its entire length to the 1,169 amino acid mSlo1 protein (3) as well as Drosophila Slo1 (dSlo1) (1, 2). The hydrophilicity profiles of both sequences indicate 11 hydrophobic segments, S0 through S10 (Refs. 3 and 27 and Fig. 1B). As with mSlo1, these can be divided into core and tail domains. Similarity between mSlo3 and mSlo1 is greatest in the core domain. The mSlo3 core (S0 through S8: mSlo3 residues 35 through 641) shares 56 and 50% identity with mSlo1 and dSlo1 cores, respectively. (Interspecies homologues mSlo1 and dSlo1 share 62% identity.) The mSlo3 tail (S9 and S10: mSlo residues 686-1136) shares 39% identity with mSlo1 and dSlo1, respectively. (The interspecies homologues mSlo1 and dSlo1 share 68% identity in this region.) A linker region having no significant conservation is found between S8 and S9. More detailed comparison of mSlo3 and mSlo1 sequences implied two functional properties of mSlo3. 1) The absence of the "calcium bowl" (18) suggested that mSlo3 may be activated by factors other than Ca2+. 2) In the K+-selective pore, a GFG motif, rather than the typical GYG, suggested differences in ionic selectivity between the two channels (28, 29).
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Heterologous Expression of mSlo3 cRNA Produces Voltage- and pH-sensitive Currents-- When expressed in the Xenopus oocyte expression system, mSlo3 cRNA produced currents that were sensitive to both pH and voltage. This was demonstrated in observations of single channel behavior (Fig. 2), macroscopic currents in the patch configuration (Fig. 3), and whole cell oocyte currents recorded in the two-electrode voltage clamp mode (Fig. 4). In patches where unitary openings could be resolved, mSlo3 single-channel currents were observed to have very brief open times. Currents were sensitive to intracellular pH. Currents were small or absent at a pHi of 7.1 or lower, whereas raising pHi resulted in sharp increases in channel activity (Fig. 2). As shown in Fig. 2, the effect of changing pH was completely and repeatedly reversible. Macroscopic currents in the inside-out patch configuration behaved consistently with single channel behavior, despite channel rundown, which occurred over the course of minutes (Fig. 3). Patches exposed to pH 7.1 produced virtually no current. However, raising the pH to 8.0 resulted in macroscopic currents that responded to depolarization and exhibited little or no inactivation. In the whole cell configuration, mSlo3 currents resembled a voltage-dependent delayed rectifier and, consistent with macroscopic currents in the patch clamp configuration, showed little or no inactivation (Fig. 4). This behavior indicates that the internal pH of oocytes must be higher than pH 7.1, consistent with previous reports that reveal resting Xenopus oocyte pH near 7.5 (30, 31). Based on this assumption, it was predicted that the manipulation of internal pH would either reduce the amplitude of observed whole cell currents (after acidification) or increase the amplitude of currents (after alkalization). Thus, acidification of the oocyte using bicarbonate-based bath solution (30) reduced the amplitude of currents (Fig. 4A, middle), whereas alkalization by ammonium chloride (31) increased the amplitude of mSlo3 currents even after attenuation by bicarbonate (Fig. 4A, right).
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Slo3 Currents Show Relaxed K+ Selectivity-- One conspicuous difference in sequence between mSlo3 and mSlo1 occurs in a region implicated in ion selectivity. All channels with high selectivity for potassium over sodium have a GYG sequence motif in the "P" region (28, 29). In contrast, mSlo3 has a GFG at this location (Fig. 1, residue 279). It was previously shown in a Shaker K+ channel that substitution of F (Phe) for Y (Tyr) in the GYG sequence decreased selectivity for K+ over Na+ (32). This suggested that mSlo3 could have less selectivity for K+ over Na+ than most potassium channels. To test this possibility, we analyzed the reversal potential for mSlo3 current tails at various concentrations of external potassium and sodium ion (Fig. 4). Our results showed that mSlo3 is less selective for K+ over Na+ than Slo1, having a PK/PNa of approximately 5 versus >50 for Slo1 (3, 5).
Slo3 Expression Is Prominent Only in Spermatocytes-- Figs. 5, A and B, show results from RT-PCR performed on RNA from brain, skeletal muscle, lung, liver, kidney, and heart; only testis produced a positive signal. Northern blots using total RNA from the same tissues gave a robust signal after only 4 h of exposure. A transcript of approximately 4 kb was seen only in testis (Fig. 5). A longer exposure (18 h) failed to reveal bands from any additional tissues (data not shown).
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A Human Slo3 Homologue Is Present in Testis--
Northern analysis
of a human tissue blot also revealed a band at 4 kb, indicating the
presence of a human Slo3 homologue of a conserved transcript size. The
human Northern blot similarly indicated a restricted tissue
distribution (Fig. 5). The small message size is unusual for a gene
encoding such a large protein. Apparently, there may be as little as
700 bp of 5- and 3
-untranslated regions. This small message size
contrasts with the larger message size of mSlo1, which is more than
twice as large in brain (3). Preliminary sequence data indicates hSlo3
is highly conserved at the molecular level.
In Situ Hybridization-- In testis, in situ hybridization revealed that mSlo3 message is expressed in the seminiferous tubules with the signal directly over developing spermatocytes (Fig. 6). The message is most abundant in the inner segments of these rings, corresponding to positions over maturing spermatocytes and possibly to early spermatids. The outermost regions of these rings contain spermatogonia, the stem cell from which spermatocytes are derived. The absence of hybridization (arrow in Fig. 6) in the peripheral areas suggests that expression is restricted to later stages of spermatogenesis. The absence of message in the interstices between rings also suggests that the nerves, blood vessels, and connective tissue in these regions of the testis probably lack expression of mSlo3.
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DISCUSSION |
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Sensitivity to both intracellular alkalization and membrane depolarization distinguishes mSlo3 from other cloned channels. Previous reports demonstrate that other channels from various tissues are sensitive to pHi, but their functional properties as well as their tissue distributions differ from mSlo3. One example is the acid-sensing channel (ASIC), which opens upon intracellular acidification (33). These channels are present in dorsal root ganglia and are involved in pain sensation. The ASIC channel is in the amiloride-sensitive Na+ channel degenerin family and is not similar to K+ channels. In contrast, TASK and RACTK1 are in the K+ channel superfamily but lack the S4 voltage sensor and do not respond to membrane potential changes. TASK is a recently cloned, four-transmembrane domain channel responsive to extracellular proton concentrations in the physiological range; it is presumed that the TASK channel contributes to the resting K+ conductance (34). The RACTK1 kidney K+ channel, a two transmembrane domain channel, is activated by intracellular alkalization but is voltage-insensitive (35). In contrast, mSlo3 as well as other Slo family channels may have evolved to respond to both voltage as well as other intracellular factors; calcium in the case of mSlo1 and pH in the case of mSlo3.
The properties of mSlo3 make it difficult to work with. In addition to
its very short mean open time and low open probability (Po) at physiological voltages, there is also a
nagging rundown problem to contend with. It is especially difficult to get patches with sufficient channels for macroscopic current analysis. Because very large depolarizations are required to elicit activity of
the channel, g/gmax plots do not
approach saturation and the Boltzmann fitted to these plots can only
serve as an estimate of channel behavior. However, from these
approximate Boltzmanns, the whole-cell current
V50 appears to be +72 ± 4.9 mV (mean ± S.E.; n = 12; Fig. 3C). The slope of
these V versus g/gmax
relations is extremely shallow (approximately 16 mV/e-fold). Thus, the
base of the curve is in a physiological range even though
gmax is not attained until +200 mV. Because of this shallow
slope, it appears that at least some channels must be open in the
physiologically relevant voltage range. Perhaps the mSlo3 channel is
designed to provide small increments of current. Alternatively, our
heterologous expression system might be missing something that affects
gating properties, perhaps a cytoplasmic factor or auxiliary subunit native to sperm that contributes to the gating of this channel. One
hint that cytoplasmic factors may be important is the fact that the
estimates of V50 from whole cell voltage clamp
recordings are shifted approximately 25 mV relative to the
macroscopic patch current (Fig. 3C). This may suggest that
another factor does indeed influence channel gating. A missing factor
could permit the mSlo3 channel to function more prominently within the
pH and voltage range present in spermatocytes or mature sperm.
The mechanism of proton sensitivity in mSlo3 is unknown. In ROMK inward rectifier channels, a titratable lysine residue has been shown to confer reversible proton block when pH is reduced below 7.5, both in whole cell recordings from oocytes exposed to bicarbonate-containing media or in perfused detached patches (30, 36). It is possible that mSlo3 is inhibited by protons by a similar mechanism. In Slo1 channels, raising [Ca2+]i increases channel activity by enabling the channel to activate at more hyperpolarized voltages (8, 9). Preliminary data from mSlo3 suggests that changing the proton concentration may not alter the voltage range of activation of the channels. Although Slo1 Ca2+ sensitivity is altered at different pHi, this effect may depend on the alteration of calcium affinity by protonating residues involved in calcium binding (37). It is not clear if or how this may relate to mSlo3 gating.
mSlo3 represents a new member of the Slo family of channels. Surprisingly, although these channels share a great deal of sequence identity, the functional features of these channels, exceptionally large conductance, calcium sensitivity, and high selectivity for K+ over Na+, do not appear to be highly conserved Slo family characteristics. The structure of the Slo1 channel resembles a voltage-dependent channel core with an appended tail region that plays a modulatory role in gating by sensing Ca2+ (17, 18). We previously demonstrated the existence of two calcium sensing sites in the mSlo1 channel, the calcium bowl, a high affinity site in the tail region, and a lower affinity site at a location that has not yet been determined (18). The lack of Ca2+ sensitivity in mSlo3 implies that neither of these two sites are present. Properties associated with the core domain also differ between Slo3 and Slo1. Slo3 is approximately 350-fold less sensitive to external TEA. Underlying this difference may be the absence of a tyrosine residue at a site reported to be critical to external TEA sensitivity (38). Rather than a tyrosine residue as in TEA-sensitive channels, the mSlo3 sequence has a valine at the corresponding position (valine at position 283). The highly selective toxins that block BK channels, charybdotoxin (39) and iberiotoxin (40), did not affect mSlo3 currents at 50 and 20 nM, respectively (data not shown). In contrast, an S4 voltage-sensing domain is present in both Slo1 and Slo3, corresponding to voltage sensitivity, which the channels share (41). The pairing of voltage sensitivity with sensitivity to a variety of intracellular factors may be the unifying feature of the Slo family.
Several observations indicate uniquely abundant expression in testis: a high density of labeling in in situ experiments, a strong signal in Northern analysis using total RNA, and high representation in a testis cDNA library ("Materials and Methods"). These data cannot exclude very low levels of expression or spatially restricted patterns of expression within the other tissues examined. However, if mSlo3 expression is indeed largely restricted to spermatocytes, it may be that the pairing of sensitivity to pH and voltage is designed to fulfill a unique role in spermatocytes. All proteins utilized by the mature sperm are synthesized during spermatogenesis, as mature sperm lack translational activity. Thus, although the mSlo3 protein has not yet been identified in mature sperm, robust transcription in developing spermatocytes makes it likely that the channel is present at these later stages. The unlikely alternative is that mSlo3 is utilized only during a narrow window of time in sperm development. Assuming its presence in mature sperm, the unusually high permeability ratio of sodium to potassium could allow multiple roles for mSlo3 channels that depend on the extracellular environment that sperm encounter. In high external Na+ or K+, Slo3 channels could have a depolarizing influence; conversely, where these ions were low in the external environment, the open channel would be hyperpolarizing. It is intriguing to speculate on the role of mSlo3, since both alkalization and depolarization are components of the signaling pathway during both sperm capacitation and the acrosome reaction, two essential steps preceding sperm fertilization of the oocyte (21, 42-45). Between mating and fertilization, sperm undergo capacitation, a process that later enables them to fertilize the oocyte. Capacitation involves an increase in cytosolic pH (pHi), which promotes metabolic and swimming activity (42, 46-47). An increase in pHi, changes in membrane potential, and a rise in cytoplasmic [Ca2+] trigger the acrosome reaction upon contact with the oocyte (21, 43, 48). As the sperm membrane depolarizes, voltage-gated calcium channels open, permitting the entry of calcium and thereby triggering the release of the acrosomal granule (23). Although the role of Slo3 in these processes remains speculative at this time, it is plausible that this channel plays a role in coordinating these events by directly linking cellular pH and membrane voltage.
Future investigation may focus on substances known to affect sperm function, which may use this channel as a target. Finally, agents that block or open this channel may be useful in the study or control of fertility.
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ACKNOWLEDGEMENTS |
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We thank gratefully Ray Gerfen and Dr.
William Snider, Washington University School of Medicine (WUSM), for
performing in situ hybridization and Dr. Graeme Mardon
(Baylor College of Medicine) for providing the testis cDNA library.
Dr. David Chaplin (WUSM), Dr. Karen O'Malley (WUSM), and Dr. P. K. Wagoner (Icagen) provided WEHI-3 library, -actin primers, and the
human tissue blot, respectively. Dr. Chris Silvia (Icagen) provided
human Slo3 sequence data. Drs. Donner Babcock and Bertil Hille
(University of Washington, Seattle, WA), Dr. Harvey Florman (Tufts
University), and Drs. Celia Santi and Alberto Darszon (Universidad
Nacional Autónoma de México de Mexico, Mexico City)
provided valuable insights.
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FOOTNOTES |
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* Supported by grants from the National Institutes of Health and Muscular Dystrophy Association (to L. S.) and the I. Jerome Flance Medical Scientist Traineeship of the Edison Foundation (to M. Schreiber).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039213.
¶ To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, Box 8108, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: salkoffl{at}thalamus.wustl.edu.
1
The abbreviations used are: mSlo3, mouse Slo3;
RT-PCR, reverse transcription-polymerase chain reaction; MOPS,
4-morpholinepropanesulfonic acid; DIDS,
4,4-diisothiocyanostilbene-2,2
-disulfonic acid; TEA,
tetraethylammonium; bp, base pairs(s); kb, kilobase(s).
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REFERENCES |
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