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Calcium-activated potassium channels expressed from cloned complementary DNAs
Neuron,
Vol.
9, 209-216,
August,
1992,
Copyright
0
1992
by Cell
Press
Calcium-Activated Potassium Channels Expressed from Cloned Complementary DNAs John P. Adelman, Ke-Zhong Shen, Michael P. Kavanaugh, Robin A. Warren, Yan-Na Wu, Armando Lagrutta, Chris T. Bond, and R. Alan North Vellum Institute Oregon Health Sciences University Portland, Oregon 97201
Summary Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the s/o locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteinsof about 1200 aa. The predicted sequences of these proteins differ by thesubstitution of blocksof aminoacids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The singlechannel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different subsituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels. Introduction Calcium-activated potassium channels exist in many cell types, including erythrocytes (Gardos, 1958), neurons (Meech, 1974; Krnjevit and Lisiewicz, 1972), muscle (Pallotta et al., 1981), hepatocytes (Jenkinson et al., 1983), and endocrine (Marty, 1981) and exocrine (Petersen and Maruyama, 1984) cells. Voltage-dependent and -independent classes of channels are distinguished, with many subtypes in each class (reviewed by Blatz and Magleby, 1987; Ewald and Levitan, 1987; LaTorre et al., 1989; Marty, 1989; Haylett and Jenkinson, 1990). The voltage-dependent calcium-activated potassium channels in excitable tissues take part in action potential repolarization (Adams et al., 1982; Elkins et al., 1986) and/or determine the rate of firing (Gorman and Thomas, 1978; Smith and Thompson, 1987), whereas in gland cells the channels are critical in the regulation of secretion (Petersen and Maruyama, 1984; Marty, 1989). These channels usually have a large unit conductance (>I00 pS) and have been referred to as maxi K or big K (BK) channels (LaTorre and Miller, 1983; Blatz and Magleby, 1987; Marty, 1989). In Drosophila flight muscle, a calcium-activated po-
tassium current contributes to action potential repolarization (Elkins et al., 1986). This potassium current is absent in flies that carry homozygous mutant slowpoke (s/o) alleles (Elkins et al., 1986; Gho and Mallart, 1986; Singh and Wu, 1989; Komatsu et al., 1990; Saito and Wu, 1991). Ganetzky and colleagues recently isolated a partial s/o cDNA (Atkinson et al., 1991). The predicted translation product contained a sequence of amino acids that closely resembles the region thought to line the ionic pore, which is conserved in all members of the Shaker-like potassium channel families (Guy and Conti, 1990; Hartmann et al., 1991; MacKinnon and Yellen, 1990; Yellen et al., 1991; Yool and Schwarz, 1991; Kavanaugh et al., 1991, 1992). These results led to the suggestion that the Slo protein might be a structural component of a calcium-activated potassium channel (Atkinson et al., 1991). In this paper, we report the coding sequences of a large family of alternatively spliced s/o transcripts obtained from analysis of s/o cDNAs. mRNA transcribed in vitro and injected into Xenopus oocytes directed expression of large conductance potassium channels that aregated both by membrane potential and by intracellular calcium concentration ([Ca2+],). Results A Family of Alternatively Spliced cDNAs Based upon the predicted partial Slo amino acid sequence (see Experimental Procedures), two degenerate oligonucleotide chains were synthesized and employed in the polymerase chain reaction (PCR) using as substrate cDNA reverse transcribed from adult fly head RNA. The reaction yielded a single band of the expected size, and DNA sequence analysis confirmed the identity of the product. Based on this sequence, a unique oligonucleotide was synthesized and used as a hybridization probe on a fly head cDNA library; 46 hybridizing clones were purified and subjected to further study. Restriction and DNA sequence analysis showed that all clones were derived from the s/o locus and contained the probe sequence, although all retained at least one apparent intron. The first 15 clones analyzed had the same N-terminal coding sequences, with an apparent intron preceding the open reading frame and that appeared to lack an AUG translation initiation codon (see Atkinson et al., 1991). To determinewhether any clones contained a different configuration at the Send, the PCR was performed on each clone using oligonucleotides that would amplify the known Sconfiguration; one chain was directed to the presumed intronic sequence 5’ to the known coding region and the other to a sequence within the coding region. One clone (SP693-4) did not amplify, and DNA sequencing revealed a 17 aa extension to the N-terminal coding sequence, in frame with the rest of the protein. The open reading frame begins with an AUG
Figure
1. The N-Terminal
Region
of Slo
The first 334 residues are shown, aligned in the hydrophobic (S156) and pore-forming regions with sequences from Shaker, Shab, to improve .!%a/, and Shaw. Dashes indicate a gap introduced alignment. Asterisks indicate identical residues or conservative substitutions.
codon within a perfect consensus sequence for Drosophila translation initiation (Cavener, 1987). Except for intron interruptions, all clones encoded the same amino acid sequence for residues 18-326; these are predicted from hydrophobicity analysis to include the first five and part of the sixth membranespanning domains (Sl-S6; Figure 1). In contrast tothe N-terminal region, thecentral portion of the molecule (within the C-terminal putative intracellular domain; residues 327-797) exhibited considerable diversity (Figure 2). In this region, the first 5 clones analyzed contained several alternative exon combinations with no clone exactly the same as any other. To determine the nature and extent of alternative coding combinations, oligonucleotides directed to sequences flanking the alternative regions were employed in PCRs using fly head cDNA as substrate. DNA sequence analysis of the resulting PCR product revealed a strikingly complex mixture of exon combinations. The coding mosaic of this domain is presented in Figure 2. The first alternative segment begins within S6 and extends for 30 aa; we have identified three distinct alternative sequences in this position (Al-A3). Following 37 conserved residues (the B domain), a stretch of 35 aa can be either of two sequences (Cl or C2), and this is followed by 104conserved amino acids (the D domain). This is followed in turn by either El or E2, each 37 aa in length, and then by 79 conserved residues (the F domain). Six sequences of different
lengths were found in the next alternative module, the G domain (13,22,27,47,59, or 60 aa); the Cl and G4 sequences were reported previously (Atkinson et al., 1991). The H region consists of 65 conserved amino acids and contains a site resembling the ATP-binding region of tyrosine kinases (Kamps et al., 1984; see Atkinson et al., 1991). This is followed by the I region, 22 aa that may or may not be present (see also Atkinson et al., 1991). The J region (440 aa) comprises the rest of the coding sequence; several further sets of oligonucleotides were employed in PCRs to determine whether the J region contains variable domains, but no alternative sequences were identified. The longest cDNA open reading frame predicts a Slo protein of 1235 aa. Thus, a conserved N-terminal domain, containing almost all of the six probable transmembrane domains, is followed by a series of alternative yet related modules. This analysis predicts up to 144 different coding combinations. To determine which combinations could be readily detected in fly head mRNA, 102 Ml3 subclones derived from the PCR reaction were probed as dot blots using separate oligonucleotide probes for each of the alternative domains; 23 distinct combinations were cataloged. Furthermore, DNA sequence analysis of clones isolated from the fly head cDNA library provided 8 additional coding combinations. Neither analysis provided evidence for restricted combinations of alternative exons.
Functional Expression of Calcium-Activated Potassium Channels Membrane patches from oocytes that had been injected more than 3 days before with RNA transcribed from a s/o cDNA (Al/C2/E2/G5/10 unless otherwise stated; Figure 2) showed unitary currents of large amplitude when calcium was applied to the cytoplasmic side (Figure 3). The amplitude of the currents was linearly dependent on the holding potential and reversed at zero potential when the potassium concentration was equal (120 mM) on both sides of the membrane; the conductance was 123 it 6.6 pS (n = 6) (Figure 3). When the potassium concentration was changed on one or other side of the membrane, the unitary currents were well fitted by the constant field expression, I = P. F?. V. [[K’ln - [K+],exp[-VFiRqjlRT where F and R have their usual il - exp[-VFiRT]), meaning, [K’]” is the extracellular K’ concentration, and T is 293OK. For the three conditions (2.5 m M [K+],,, 120 m M [K+],; 120 m M [K’],, 120 m M [K’],; and 120 m M [K+],, 2.5 m M [K’],) the estimates for P were 2.6, 2.7, and 3.5 x IO-‘? cm3/s, respectively; the extrapolated zero current potentials were close to -lOO,O, and 100 mV, respectively, which are similar to the theoretical values for the potassium equilibrium potential. At a given [CaZ+],, the probability of opening of the channels was strongly voltage dependent. At 100 PM [Ca”], very brief openings occurred extremely rarely at -80 mV (Figure 3A); depolarization increased both
Clontng
and Expression
of gic,
211
Figure 2. The Central gion of Slo
/
:AACAGCCAAA
Re-
(A) Schematic depiction. Alternative forms of the same length (A, C, and E) are boxed. Alternative forms of variable length (C and I) are also indicated. Numbers refer to the number of amino acids in each region. (B) Amino acid sequence of Slo (315-1235). Sequences in the C region are presented with gaps to achieve alignment. Variants within the regions A, C, E, and C have been numbered from top to bottom. Underline indicates sequence with possible resemblance to calcium-binding domain.
\
/
and C-Terminal
S’ M Ax Ga ’ S,Ml
B
01-6 646
CKN
LATFRKGVRAVQMVGRAKDDEYSLSNEHHPAPTFTPPELPKRVHVRGSVSGDITRDRED’r I.ATFRKGVRAV;MVGRA.................................SDITRDR~DT
NL
I,T”QPRSKFDDLV............................................... I,TVQPRSKFDDL.............“~HHPAPTFTPPELPKRVHVRGSVSG”~TRDREDT I,T”QPRSKFDDI,......................................GDITRDREDT I)VGMTMMQTGMVNQGITSVMNTMF: DEHHPAPTFTPPELPKRVHVRGSVSGD~TRDR~~~T 111
LNRN’JR~I’N~~GNGTGGMHHMNNTAAAAAAAAAAAGKQVNKVK~jTV~SRQV~GQV~SPSQY~R
77 i
IO, 1 NlilDANPYAGYQLAYEVKKIIM
PTSKSSGTGTQNQNGG”SLPAGIADDOSKDFDFEKTEMKYDSTGk,b
847
HWSPAKSLEDCILI~RNQAAMTVl,NGHV\“V’CI,FnDPDSPI~GS
901
‘~I)YIRREirlKMLQNL.PKISVLNGSPLSHADLHRVNVT,.ADK~A~L,ASLN
916
I KIIM’~F’I,DTlG”I,SQHGPEFDNI.SATAOSFIVL~~DP
1043
DT~:I.‘/I.‘I’V~FACGT~FA”S’~~,DSL~ST’~YF~QNA~~,,IKSL1
1110 117
I’PE
I’GGA~I’CLCI.I1,REGAGLRGGYSTV
~:SI,SNK~~RCR\IGQTSI,~DPLA~~G~~~:K~G~~L~.'~A*LKSYG~L~IC:LYR~‘RL)I'SSSCDA~SKR~V~ /
'PN~'P"DPS~,~,PTD(,VFVL.M()FDPGLEYKPPAV~APAGC:RGTN',~GSGVGGGCSNKUDNS
the frequency of openings and the mean open time (Figure 3). Ensemble currents activated exponentially, becoming faster with stronger depolarization (Figure 4C). At 300 uM [Ca2+],, time constants were 3.9 + 0.31 ms, 3.4 & 0.30 ms, 2.7 k 0.17 ms, 2.6 + 0.12 ms, and 2.1 f 0.13 ms at +60 mV, +80 mV, +I00 mV, +I20 mV, and +I40 mV, respectively (n = 3-6). The current declined by less than 10% during depolarizations continued for 5-10 s (30 uM or 100 uM [Ca2+],, +80 mV, n = 5). Channel activity was absolutely dependent on [Ca2+],, even at very positive test potentials (+80 mV). At a given potential, the current increased with [Ca”], in the range l-100 PM (Figure 4), the most striking effect being to shift negatively the voltage at which the cu:rent activated (Figures 48 and 4D). The time
1235
course of activation of the current also became more rapid in higher [Caz’], (Figure 4C). At +80 mV, activation time constants were 27.0 + 1.2 ms, 13.8 + 1.6 ms, 4.0 f 0.2 ms, and 2.1 f 0.13 ms at 3 PM, IO PM, 100 PM, and 300 PM [Ca”],, respectively (n = 3-6). Many combinations of substituted blocks of amino acids in the A, C, E, G, and I domains are predicted from the isolated cDNAs. The functional properties of A1/C21E1/G3/10 were compared with those of Al/W E2/G5/10. There was no difference in the unitary conductance. However, at a given [CaZ’l, and potential, the channel openings of Al/C2/El/G3/10 were clearly much longer than those of Al/C2/E2/G5/10 (Figure 5). At 60 mVand 30 uM [Ca2+],, theopen timedistributions were fit by two exponentials (Figure 5). The time constant of the longer component was 97.1 f 16.1 ms
Neuron 212
Figure 3. Currents Channels
86 25 65 c4 E $3
80
s2 21 60 40-
OO 0
20M -20 “r”“m ”r”“““T””
10 20 Amplitude (PA)
30
c
-4oc -607
V W)
100lM 1
d---c
10vM
-
3pM
-
IIOPA
(A) Records of channel activity at different potentials (indicated beside each trace, mV). [Ca2’], was 100 PM, and potassium concentration on both sides of the membranepatchwas120mM.(B)Amplitudedistribution from a recording such as shown in (A) (at f60 mV). This is an all-points histogram (sampled at 1 kHz); the baseline noise is not shown (broken line). (0 Currentvoltage relation for unitary currents. Open circles: [K+], (bath) and [K+], (pipette) were both 120 mM. Closed circles: [K’], = 120 mM; [K’],, = 2.5 mM. Closed squares: [K’], = 2.5 mM; [K+],, = 120 mM. The mean current amplitude at each potential was determined from the amplitude distribution at that potential; points shown are means of these values from three to six patches, with SEM where this exceeded the size of the symbol.
Figure 4. Calcium tassium Currents
B
NPM
-50
s/o Potassium
potassium conductance in some tissues (Haylett and Jenkinson, 1990); neither affected s/o currents in oocytes (at +80 mV, 30 PM [Ca*+],, control: 9.0 + 1.0 nS, charybdotoxin [I uM]: 8.8 + 1.3 nS, n = 4; control: 3.4 f 0.24 nS, apamin [3 KM]: 3.1 f 0.27 nS, n = 3). On the other hand, tetraethylammonium (TEA) was a very potent channel blocker at the extracellular side (added to the pipette solution). At 100 uM TEA, openings of single channels were interrupted by many flickery closures; at 1 m M the unitary current traces were noisy and of reduced amplitude; and at IO m M unitary currents were not seen. TEA (100 PM) reduced
(n = 6) for Al/C2/El/G5/10 and 6.7 f 1.6 ms (n = 11) for Al/C2/E2/C5/10. Further systematic experiments are in progress to determine the contributions of the all the variable regions of the molecule to the gating kinetics and calcium sensitivity. A putative calcium-binding region is located within El (Figure 2B) (Atkinson et al., 1991); we expressed the channel in which these 12 residues were deleted by site-directed mutagenesis of Al/C2/El/G5/10. The prop erties of the expressed channel were not obviously different from those of the wild-type Al/C2/El/G5/10. Charybdotoxin and apamin blockcalcium-activated
A
through
50 0 Potential (mV)
C
Potential (InV)
100
Dependence
of s/o Po-
(A) Channel activity recorded at f40 mV In fourdifferentcalcium concentrationsfindicated by each trace). Each sample shown IS 500 ms. (B) Open probabilities at different [Ca’+], and potential. These observations were made on a patch containing only one active channel (no double openings were observed in 5 min of recording even at 300 uM [Cal],, +60 mV). A cursor set at 50% mean amplitudewas used to measureopen CT.,) and closed (1,) durations, and open probability was computed from s,/fs,, + T,). This illustration is from Al/C2/El/G3/10. (C) A membrane patch was depolarized from -60 mV to +80 mV for200 ms; records show averagecurrents recorded from 30such depolarizations. The depolarizing steps were repeated in fivecalcium concentrations (indicated beside each trace). (D) Voltage dependence of potassium conductance measured from ensemble records such as shown in (C). Calcium concentration indicated beside each trace; 3-11 patches for each point. In all records, potassium concentrations were 2.5 m M in the pipette and 120 m M in the bath.
Cloning 213
and Expression
of gkcr
B
A
Al/C2/El/GWlO
Ai/C2/EZ/G5/10
TEA was only a very weak blocker of the potassium current when applied to the cytoplasmic surface of the patch (10 m M inhibited by 24% + 2%, n = 8; 30 m M by 52.6% + 2.3%, n = 5; +I00 mV, 30 PM or 100 uM [Ca2’],). It might be thought that the low sensitivity to internal TEA correlates with the Ser residue at position 300. Other voltage-gated potassium channels have Thr in this position, but mutagenesis to Ser reduces IO-fold the affinity for internal TEA (Yellen et al., 1991). We expressed the mutant channels in which Ser-300 was replaced by Thr or by Ala; there was no change in the sensitivity to internal TEA. Discussion
D
C Al/C2/E2/G5/10
Al/C2/Ei/G3/10
120
120,
100
100 80 M) 40 i
Figure
5. Difference
between
Two s/o Variants
(A) and (C) are from Al/C2/E2/C5/10; (B) and (D) are from Al/C2/ El/C3/10. (A and B) Sample records at +60 mV in the calcium concentrations indicated. Al/C2/E2/C5/10 showed no openings when the calcium concentration was 10 PM. (C and D) Distribution of open times for two typical patches expressing Al/C2/E2/ C5/10 and Al/C2/El/C3/10, both at 60 mV in 30 uM [Ca’+],. The distributions are fitted by Alexp(-t/T,) + A,expt-t/r,); for Al/C21 E2/G5/10 A, = 202, 7, = 0.6 ms, A2 = 73, T> = 4.3 ms and for Al/ C2/El/G3/10 A, = 84, T, = 11 ms, Ar = 65, T> = 86 ms.
the ensemble current from 884 + 134 pA (n = 9) to 489 k 71 pA (n = 5; at +I00 mV); this high sensitivity to external TEA is characteristic of voltage-dependent, calcium-activated potassium channels of high unit conductance in many different cells (LaTorre et al., 1989; Haylett and Jenkinson, 1990; Adams et al., 1982; Yellen, 1984). The effectiveness of extracellular TEA is also consistent with the presence of a Tyr residue at position 308, because Tyr in the equivalent position of other voltage-dependent channels provides a high affinity TEA-binding site (MacKinnon and Yellen, 1990; Kavanaugh et al., 1991, 1992; Heginbotham and MacKinnon, 1992). Therefore, the s/o channel was expressed in which this Tyr was mutated to Val. Unlike the normal (wild type) channel, the mutant channel remained functional in the presence of IO m M external TEA (n = 15). This result indicates that, despite the differences in amino acid sequences between s/o and the Shaker-like channels, the same residues contribute to the external mouth of the pore (Guy and Conti, 1990; Durell and Guy, 1992).
The results identify the s/o gene product as a calciumactivated, voltage-dependent potassium channel. Although there is little primary sequence homology to other cloned potassium channels, several important residues in the hydrophobic regions are the same as those in Shaker-like channels, most notably the poreforming segment (Figure 1) (Wei et al., 1990; see Atkinson et al., 1991). Our assignment of membranespanning regions Sl and S2 differs from that suggested by Atkinson et al. (1991), to provide better alignment of several key residues in these regions (Figure 1; see also Durrell and Guy, 1992). Some of the conserved residues are also found in ion channels other than potassium channels; other amino acid residues, such as the Cys residues, which occur in S2 and S6 of all previously known potassium channels and the photoreceptor cation channel (Guy and Conti, 1990; Jan and Jan, 1990), are absent in Slo. The pore-forming region of the channel (usuallydefined as the 21 residue segment in the S5-S6 domain delineated by 2 Pro residues in Shaker, Shaw, Shab, and Shal; see Figure 1; Durell and Guy, 1992) is well conserved between s/o and other voltage-dependent channels in its C-terminal one-half, though not in the N-terminal one-half. Slo has a Cys residue in the position equivalent to the second of the 2 Pro residues; we found no obvious differences in the properties of s/o channels in which this Cys was changed to Pro by site-directed mutagenesis (data not shown). Immediately adjacent to this Cys is a Tyr residue in Slo (position 308; Figure 1). The rat brain potassium channel RBKI, which is a member of the Shaker family cloned from rat brain (Christie et al., 1989), has a Tyr residue (Tyr-379) in the analogous position. Indeed, all channels having a Tyr in this position are very sensitive to block by external TEA; the only substitution that retains the high affinity is Tyr to Phe, leading to suggestion that the aromatic electrons are important for the binding of TEA (Kavanaugh et al., 1991 and references therein; see also Heginbotham and MacKinnon, 1992; Kavanaugh et al., 1992). In RBKI, the replacement of Tyr by Val reduces the TEA affinity by about 30-fold. The present finding that the slo channel was very sensitive to TEA and that this sensitivity was dramatically reduced by the mutation Y308V, indicates that
NWKNl 214
the external mouth of the s/o channel has a threedimensional structure that is fundamentally similar to that of Shaker channels (Durell and Guy, 1992). The full significance of the abundance of alternatively spliced gene products from adult fly head cDNA remains unknown. The other families of potassium channels cloned from Drosophila, most notably the Shaker channels, rely on alternative splicing to generate structural diversity. Alternatively spliced Shaker variants have been shown to result in functional differences among the expressed channels (Schwarz et al., 1988; Timpe et al., 1988; Hardie et al., 1991), and we observed marked differences in the mean channel open time between variants Al/C2/E2&5/10 and Al/ C2/El/G3/10 (Figure 5). Finally, the different splice products that have been isolated all came from adult fly head cDNA; it is quite possible that they do not represent the full complement of mRNAs transcribed from this locus, because other developmental or tissue-specific forms may exist. The properties of the single-channel currents observed in oocytes expressing s/o resemble in several respects those of thecurrent that is missing in skeletal muscle fibers of the s/o Drosophila mutant (known as 7,8). The single-channel conductance is k, ICFI or &cd; somewhat higher (123 pS versus 88 pS) but the rapid activation by depolarization is similar. It has been deduced from electrophysiological studies in Drosophila muscles and neurons that current through s/o channels inactivates during a sustained depolarization; the currents in Xenopus oocytes did not inactivate very much during several seconds. Inactivation in Drosophila muscle might be secondary to a reduction in [Ca*+],, or Drosophila channels might form as heteropolymers with other subunits that confer distinct inactivation properties, as has been found for the Shaker family of channels (Schwarz et al., 1988). As in Drosophila muscle (Komatsu et al., 1990) and mammalian cells (Marty, 1981, 1989; Blatz and Magleby, 1987), channel activity persisted for hours in excised patches; this suggests that gating by calcium does not require other cytoplasmic proteins. There is a sequence in Slo (Figure 2) that is stated by Atkinson et al. (1991) to resemble the EF hand of calciumbinding proteins (see Marsden et al., 1990); its removal by site-directed mutagenesis made no obvious difference to the properties of the expressed channel. It will be important todetermine whether other putative EF-binding domains exist that are critical to calcium sensitivity. However, an important difference between the properties of the s/o channels in Xenopus oocytes and calcium-activated potassium currents in most other cells, including Drosophila muscle, is the tower sensitivity to [Ca?‘], in the oocytes. It is possible that a higher sensitivity to [Caz+], might be found for other splice variants that have not yet been expressed; it is also possible that these channels normally form as heteropolymers and that other, yet unknown, subunits contribute to the higher affinity for [Cal+],. s/o channels might also be insensitive to [Cai’], be-
cause they undergo posttranslational modifications in most cells, which do not occur in the oocyte. For example, the calcium sensitivity of potassium channels can be markedly altered by phosphorylation with A kinase (Ewald et al., 1985; Reinhart et al., 1991; Kume et al., 1989) or possibly by autophosphorylation (Chung et al., 1991). Slo has several amino acid sequences resembling those commonly phosphorylated by kinases, including a consensus sequence for A kinase (Ser-1013). The heterologous expression of this family of channels should make it possible to examine directly the role of such processes in the function of calcium-activated potassium channels. We anticipate that the cloning and expression of similar molecules will aid in clarifying the structural and functional relationships among the members of this protean channel family. Experimental
Procedures
Isolation of cDNAs Total RNA (0.4 mg) was extracted from 2 g of fly heads (Canton-5; courtesy of Dr. M. Forte) byguanidinium isothiocyanate and acid phenol extraction; 2 ug was reverse transcribed as described (Bond et al., 1991). Two sets of degenerate oligonucleotides, AK and B/D (synthesized on an Applied Biosystems 390A PCR Mate, Foster City, CA), were employed in nested PCR. The sequences were based on a predicted fragment of Slo amino acid sequence (Robertson et al., 1990, Sot. Neurosci., abstract). In the first reaction, the A and C chains (A = GCX(CT)TX(CA)CXTT(AG)ATGAC + CCX(CT)TX(CA)GXCTXATCAC; C = CC(AC)TAXCCXACAGT(AG)CTCAT + CC(AG)TAXCCXACXGT(AG)CTCAT) were used to amplify 5% of the reverse transcriptase reaction (IO cycles: 96’C for 30 s, 45OC for 30 s, 72°C for 30 s). One-tenth of this material was transferred to a fresh tube, and a new reaction was performed with B (CGACGAATTCCCXGA(TC)AT(ATC)TT(AG)CA (AC)TA + CGAGGAATTCCCXGA(TC)ATfATC)TToCA(AG)TA) and D tGCGCAACCTTA(AC)(AG)AAfAG)TAXACfAG)CAXGT) (40 cycles: 96OC for 30 s, 55OC for 30 s, 7Z°C for 30 s). All PCRs were performed using Replinase (NEN-DU Pont, Boston, MA) on a thermocycler (Model 50, Coy Laboratory Products, Grass Lake, MI). The resulting band was cut at the artificial restrictton sites introduced by the primers and subcloned into M 13 phage, and the nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using Sequenasr (US Biochemical, Cleveland, OH). Based on this DNA sequence, a unique oligonucleotide was synthesized (CCGGGCATTATACA TCTGCTGGAGAACTCTGGC), radiolabeled at the 5’ end with 74 polynurleotide kinase (BRL, Gaithersburg, MD), and used as hybridiration probe to screen 750,000 fly head cDNA clones rn kgtll (Papazian et al., 1987; provided by Dr. M. Forte) using Gene Screen membranes (NEN-DU Pant). DNA was prepared from plaque-pure phage; the inserts were subcloned in both onentations into Ml3 phage; and the nucleotide sequences were determined. The previously described s/o cDNA sequence did not include an initiator codon (Atkinson et al., 1991). The PCR was used to determine whether any of the 46 clones isolated from the fly head cDNA library contained sequences that predicted an extended N-terminal coding sequence. Pure plaques of each of the 46 clones were excised from agar plates, and phage DNA was eluted in IO ul ofwater; 1 ~1 was used in the PCR with chains that would amplify the known 5’ configuration. One chain was dtrected within the coding region (3’ chain; CAAAGCACCAC TACTTCCGCA), and the other was directed within the known, presumably introntc, sequence 5’ to the open readmg frame ti’ chain; CAACATTGGCGAGCCGCAGCG). Following amplification (40 cycles: 96OC for 30 s, 55OC for 30 s, 72OC for 30 $1, the reaction products were visualized on an ethidium bromide stained agarose gel. Under these conditions, 1 clone fhSP693-4)
Cloning 215
and Expression
of gKcr
did not amplify, suggesting that it contained adifferent sequence in this region. Analysis of s/o cDNA clones revealed alternative exon combinations in regions A, C, E, and G. This was studied in further detail by PCR on fly head cDNA. One primer (CATCCACCTCCATACGCTTGG) was directed to the N-terminal region between S4 and S5, and the other primer (G~G~CATGTCGTGCATGCC) was directed to an invariant part of the carboxyl terminus. Following 45 cycles (96OC for 30 s, 60°C for 30 s, 72OC for 30 S) the DNA was cut with Sphl and Sacl. Bands approximately 1350 bp in length were gel purified and cloned into Ml3 phage; nucleotide sequences were obtained as described above. This strategy was repeated twice with chains directed to other regions of the carboxyl terminus (5’: GTACGCCTGGCATGCACCAC; 3’: CCACGAATrCGTGCCGCAACTGCCACTCCTCGTC and 5’: GCACGAATrCAAGTGATATCGCCATCG; 3’: GCACAAGCTTGTACGAATAAGGACCATCAG). The transcription vector used for expression (pS-) was derived from pSelect (Promega, Madison, WI) by reversing the orientation of the fl replication origin. All in vitro transcription properties of pS are identical to those of pSelect. Two plasmids were used for oocyte expression (pSPexpAlC2E2G510 and pSPexpAlC2ElG310). Both contained the 900 bp Bglll-Sac1 fragment from 1SP693-4. encoding 170 bpof Suntranslated sequences, the initiator codon, and the first four likely transmembrane segments; pSPexpAlC2E2G.510 was constructed by ligating onto this a SaclKpnl fragment from one of the hybridizing h clones (hSP3-2) that contained the rest of the coding sequence and approximately 500 bp of 3’ untranslated sequence (in which resides a unique Sal1 restriction site). To construct pSPexpAlC2ElG310, a 1330 bp Sacl-Sphl fragment generated by PCR replaced the corresponding fragment from pSPexpAlC2E2G510. To ensure that PCR mistakes were not incorporated into plasmids used for expression studies, the nucleotide sequences for each region were determined to be identical to multiple representatives isolated from the fly head cDNA library. The complete nucleotide sequences of the coding regions for all expression plasmids were verified prior to RNA synthesis and injection. Expression plasmids were linearized at the Sal1 site, and capped mRNAs were syntheisized in vitro usingT7RNA polymerase. All molecular biology enzymes were purchased from BRL. Nucleotide and protein sequence computer analysis was performed using Genetics Computer Group software. Oocyte Expression Oocytes were injected with 50 ng of RNA in 50 nl of sterile water. Inside-out membrane patches were pulled from oocytes from which the vitelline membrane had been mechanically removed after brief (5-10 min) exposure to a hypertonic solution (220 m M aspartic acid, 220 m M N-ethylglucamine, 2 m M M&I,, 10 m M EGTA, 10 m M HEPES [pH 7.21). Except where otherwise stated, the pipette (extracellular) solution contained 2.5 m M potassium ,gluconate, 115 m M sodium gluconate, 1.8 m M calcium, and 10 m M HEPES (titrated to pH 7.2 with NaOH; final sodium concentration 119 mM), and the bath (intracellular) solution contained 116 m M potassium gluconate, 2 m M magnesium gluconate, and 10 m M HEPES (titrated to pH 7.2 with KOH; final potassium concentration 120 mM). Calcium gluconate was added in amounts expected to give the free calcium concentrations stated. These were computed from stability constants for calcium gluconate and magnesium gluconate (Dawson et al., 1969). Chloride-free !solutions were used to avoid currents through calcium-activated chloride channels endogenous to the oocyte. Electrode resislance prior to seal formation was about 2 MQ; seals weretypically ‘l-5 GQ. Calcium-dependent unitary currents were observed in oocytes from 3 to 30 days after injection with RNA, but the majority of experiments were carried out on oocytes that had been injected between 5 and 10 days previously. Single-channel currents were recorded with an Axopatch IB amplifier (front panel lilter 5 kHz) and played into an A/D converter (Axolab, Axon Instruments) at 1 kHz. Subsequent analysis was as described (Derkach et al., 1989; Shen et al., 1992). Numerical data are expressed as means ? SEM for the number of patches indicated.
Acknowledgments This work was supported by grants from the United States Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
March
31, 1992; revised
May 20, 1992
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pot-
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Elkins, T., Ganetzky, B., and Wu, C.-F. (1986) A Drosophila mutation that eliminates a calcium-dependent potassium current. Proc. Natl. Acad. Sci. USA 83, 8415-8419. Ewald, D. A., and Levitan, I. B. (1987). Ion channels regulated by calcium. In Neuromodulation: The Biochemical Control of Neuronal Excitability, L. K. Kaczmarek and I. B. ILevitan. eds. (New York: Oxford University Press), pp. 138-158. Ewald, D. A., Williams, A., and Levitan, I. B. (1985). Modulation of sing1 Ca2+-dependent K+-channel activity by protein phosphoryla eion. Nature 375, 503-505. Gardos, G. (1958). The function meabilityof human erythrocytes. 654.
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Gho, M., and Mallart, A. (1986). Two distinct calcium-activated potassium currents in larval muscle fibers of Drosophila melanogaster. Pfltigers Arch. 407, 526-533. Gorman, A. L. F., and Hermann, A. J. (19791. internal effects of divalent cations on potassium permeability/ in molluscan neurones. J. Physiol. (Land.) 296, 393-410. Gorman, A. L. F., and Thomas, M. V. (1978). Changes in the intracellular concentration of free calcium ions in a pace-maker neurone, measured with the metallochromlc indicator dyearsenazo III. J. Physiol. (Land.) 275, 357-376.
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Reinhart, P. H., Chung, S., Martin, B. L., Brautigan, D. L., and Levitan, I. B. (1991). Modulation of calcium-activated potassium channels from rat brain by protein kinase A and phosphatase 2A. J. Neurosci. 17, 1627-1635.
Haylett, D. G., and Jenkinson, D. H. (1990). Calcium-activated potassium channels. In Potassium Channels, N. S. Cook, ed. (New York: John Wiley & Sons, Inc.) pp. 70-95.
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R. (1992). The aromatic bindion on potassium channels.
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Isacoff, E. Y., Jan, Y. N., and Jan, L. Y. (1990). Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes. Nature 345, 530-534.
Saito, M., and Wu, C.-F. (1991). Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts. J. Neurosci. 77, 2135-2150.
Heginbotham, L., and MacKinnon, ing site for tetraethylammonium Neuron 8, 483-491.
Jan, L. Y., and Jan, Y. N. (1990). A superfamily Nature 345, 672.
of ion channels.
Jenkinson, D. H., Haylett, D. C., and Cook, N. S. (1983). Calciumactivated potassium channels in liver cells. Cell Calcium 4, 429437. Kamps, M. P., Taylor, S. S., and Sefton, B. M. (1984). Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature 310, 589-592. Kavanaugh, M. J., Busch, tion between the pore of Biol. Chem.
M. P., Varnum, M. D., Osborne, P. B., Christie, A. E., Adelman, J. P., and North, R. A. (1991). Interactetraethylammonium and amino acid residues in cloned voltage-dependent potassium channels. J. 266, 7583-7587.
Kavanaugh, M. P., Hurst, R. S.,Yakel, J.,Varnum,M. D.,Adelman, J. P., and North, R. A. (1992). Multiple subunits of a voltagedependent potassium channel contribute to the binding site for tetraethylammonium. Neuron 8, 493-497. Komatsu,A., Singh, S., Rathe, P., and Wu, C.-F. (1990). Mutational and gene dosage analysis of calcium-activated potassium channels in Drosophila: correlation of micro- and macroscopic currents. Neuron 4, 313-321. Krnjevit, K., and Lisiewicz, into spinal motoneurones.
A. (1972). Injection of calcium ions J. Physiol. (Land.) 225, 363-390.
Kume, H., Takai, A., Tokuno, A., and Tomita, T. (1989). Regulation of Cal+-dependent K’-channel activity in tracheal myocytes by phosphorylation. Nature 347, 152-154. LaTorre, R., and Miller, C. (1983). Conduction and selectivity potassium channels. J. Membr. Biol. 77, 11-30.
in
LaTorre, R., Oberhauser, A., Labarca, P., and Alvarez, 0. (1989). Varieties of calcium-activated potassium channels. Annu. Rev. Physiol. 57, 385-399. MacKinnon, R., and Yellen, C. (1990). Mutations affecting TEA blockade and ion permeation in voltage-activated K channels. Science 250, 276-279. Marsden, B. J., Shaw, G. S., and Sykes, B. D. (1990). Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments. Biochem. Cell Biol. 68, 587-601. Marty, A. (1981). Ca-dependent K channels conductance in chromaffin cell membranes. 500.
with large unitary Nature 297, 497-
Marty, A. (1989). The physiological role of calcium-dependent channels. Trends Neurosci. 72, 420-424. Meech, R. W. (1974). The sensitivity of Helix aspersa neurones injected calcium ions. J. Physiol. (Lond.) 237, 256-277.
to
Pallotta, B. S., Magleby, K. L., and Barrett, J. N. (1981). Single channel recordings of Cal+-activated K’ channels in rat muscle cell culture. Nature 293, 471-474. Papazian, D. P., Schwarz, T. L., Tempel, B. L., Jan, Y. N., and Jan, L. Y. (1987). Cloning of the genomic and complementary DNA
Sanger, F., Nicklen, S., and Coulson, ing with chain-terminating inhibitors. 74, 5463-5468.
A. R. (1977). DNA sequencProc. Natl. Acad. Sci. USA
Schwarz, T. L., Tempel, B. L., Papazian, D. M., Jan. Y. N., and Jan, L. Y. (1988). Multiple potassium-channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 337, 137-142. Shen, K.-Z., North, R. A., and Surprenant, A. (1992). Potassium channels opened by noradrenaline and other transmitters in excised membrane patches of guinea-pig submucosal neurones. J. Physiol. (Lond.) 445, 581-599. Singh, S., and Wu, C.-F. (1989). Complete tassium currents in Drosophila. Neuron
separation ot tour po2, 13251329.
Smith, S. J., andThompson, S. H. (1987). Slow membranecurrents in bursting pace-maker neurones of Tritoma. J. Physiol. (Lond.) 382, 425-448. Timpe, L., Schwarz, T., Tempel, B., Papazian, L. (1988). Expression of functional K’channels in Xenopus oocytes. Nature 337, 143-145.
D., Jan, Y., and Jan, from ShakercDNA
Wei, A., Covarrubias, M., Butler, A., Baker, K., Pak, M., and Salkoff, L. (1990). K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse. Science 248, 599-603. Yellen, G. (1984). Ion permeation K+ channels of bovine chromaffin 186.
and blockade in Cd”-activated cells. J. Gen. Physiol. 84, 157-
Yellen, G., Jurman, M. E., Abramson, T., and MacKinnon, R. (1991). Mutations affecting internal TEA blockade identify the probable pore-forming region of a K’ channel. Science 257,939942. Yool, A. J., and Schwarz, T. L. (1991). Alteration of ionic selectivity of a K channel by mutation of the H5 region. Nature 349, 700704. CenBank
Accession
Number
The nucleotide sequences of all the cDNAs have been submitted to GenBank under accession number M96840.
Vol.
9, 209-216,
August,
1992,
Copyright
0
1992
by Cell
Press
Calcium-Activated Potassium Channels Expressed from Cloned Complementary DNAs John P. Adelman, Ke-Zhong Shen, Michael P. Kavanaugh, Robin A. Warren, Yan-Na Wu, Armando Lagrutta, Chris T. Bond, and R. Alan North Vellum Institute Oregon Health Sciences University Portland, Oregon 97201
Summary Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the s/o locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteinsof about 1200 aa. The predicted sequences of these proteins differ by thesubstitution of blocksof aminoacids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The singlechannel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different subsituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels. Introduction Calcium-activated potassium channels exist in many cell types, including erythrocytes (Gardos, 1958), neurons (Meech, 1974; Krnjevit and Lisiewicz, 1972), muscle (Pallotta et al., 1981), hepatocytes (Jenkinson et al., 1983), and endocrine (Marty, 1981) and exocrine (Petersen and Maruyama, 1984) cells. Voltage-dependent and -independent classes of channels are distinguished, with many subtypes in each class (reviewed by Blatz and Magleby, 1987; Ewald and Levitan, 1987; LaTorre et al., 1989; Marty, 1989; Haylett and Jenkinson, 1990). The voltage-dependent calcium-activated potassium channels in excitable tissues take part in action potential repolarization (Adams et al., 1982; Elkins et al., 1986) and/or determine the rate of firing (Gorman and Thomas, 1978; Smith and Thompson, 1987), whereas in gland cells the channels are critical in the regulation of secretion (Petersen and Maruyama, 1984; Marty, 1989). These channels usually have a large unit conductance (>I00 pS) and have been referred to as maxi K or big K (BK) channels (LaTorre and Miller, 1983; Blatz and Magleby, 1987; Marty, 1989). In Drosophila flight muscle, a calcium-activated po-
tassium current contributes to action potential repolarization (Elkins et al., 1986). This potassium current is absent in flies that carry homozygous mutant slowpoke (s/o) alleles (Elkins et al., 1986; Gho and Mallart, 1986; Singh and Wu, 1989; Komatsu et al., 1990; Saito and Wu, 1991). Ganetzky and colleagues recently isolated a partial s/o cDNA (Atkinson et al., 1991). The predicted translation product contained a sequence of amino acids that closely resembles the region thought to line the ionic pore, which is conserved in all members of the Shaker-like potassium channel families (Guy and Conti, 1990; Hartmann et al., 1991; MacKinnon and Yellen, 1990; Yellen et al., 1991; Yool and Schwarz, 1991; Kavanaugh et al., 1991, 1992). These results led to the suggestion that the Slo protein might be a structural component of a calcium-activated potassium channel (Atkinson et al., 1991). In this paper, we report the coding sequences of a large family of alternatively spliced s/o transcripts obtained from analysis of s/o cDNAs. mRNA transcribed in vitro and injected into Xenopus oocytes directed expression of large conductance potassium channels that aregated both by membrane potential and by intracellular calcium concentration ([Ca2+],). Results A Family of Alternatively Spliced cDNAs Based upon the predicted partial Slo amino acid sequence (see Experimental Procedures), two degenerate oligonucleotide chains were synthesized and employed in the polymerase chain reaction (PCR) using as substrate cDNA reverse transcribed from adult fly head RNA. The reaction yielded a single band of the expected size, and DNA sequence analysis confirmed the identity of the product. Based on this sequence, a unique oligonucleotide was synthesized and used as a hybridization probe on a fly head cDNA library; 46 hybridizing clones were purified and subjected to further study. Restriction and DNA sequence analysis showed that all clones were derived from the s/o locus and contained the probe sequence, although all retained at least one apparent intron. The first 15 clones analyzed had the same N-terminal coding sequences, with an apparent intron preceding the open reading frame and that appeared to lack an AUG translation initiation codon (see Atkinson et al., 1991). To determinewhether any clones contained a different configuration at the Send, the PCR was performed on each clone using oligonucleotides that would amplify the known Sconfiguration; one chain was directed to the presumed intronic sequence 5’ to the known coding region and the other to a sequence within the coding region. One clone (SP693-4) did not amplify, and DNA sequencing revealed a 17 aa extension to the N-terminal coding sequence, in frame with the rest of the protein. The open reading frame begins with an AUG
Figure
1. The N-Terminal
Region
of Slo
The first 334 residues are shown, aligned in the hydrophobic (S156) and pore-forming regions with sequences from Shaker, Shab, to improve .!%a/, and Shaw. Dashes indicate a gap introduced alignment. Asterisks indicate identical residues or conservative substitutions.
codon within a perfect consensus sequence for Drosophila translation initiation (Cavener, 1987). Except for intron interruptions, all clones encoded the same amino acid sequence for residues 18-326; these are predicted from hydrophobicity analysis to include the first five and part of the sixth membranespanning domains (Sl-S6; Figure 1). In contrast tothe N-terminal region, thecentral portion of the molecule (within the C-terminal putative intracellular domain; residues 327-797) exhibited considerable diversity (Figure 2). In this region, the first 5 clones analyzed contained several alternative exon combinations with no clone exactly the same as any other. To determine the nature and extent of alternative coding combinations, oligonucleotides directed to sequences flanking the alternative regions were employed in PCRs using fly head cDNA as substrate. DNA sequence analysis of the resulting PCR product revealed a strikingly complex mixture of exon combinations. The coding mosaic of this domain is presented in Figure 2. The first alternative segment begins within S6 and extends for 30 aa; we have identified three distinct alternative sequences in this position (Al-A3). Following 37 conserved residues (the B domain), a stretch of 35 aa can be either of two sequences (Cl or C2), and this is followed by 104conserved amino acids (the D domain). This is followed in turn by either El or E2, each 37 aa in length, and then by 79 conserved residues (the F domain). Six sequences of different
lengths were found in the next alternative module, the G domain (13,22,27,47,59, or 60 aa); the Cl and G4 sequences were reported previously (Atkinson et al., 1991). The H region consists of 65 conserved amino acids and contains a site resembling the ATP-binding region of tyrosine kinases (Kamps et al., 1984; see Atkinson et al., 1991). This is followed by the I region, 22 aa that may or may not be present (see also Atkinson et al., 1991). The J region (440 aa) comprises the rest of the coding sequence; several further sets of oligonucleotides were employed in PCRs to determine whether the J region contains variable domains, but no alternative sequences were identified. The longest cDNA open reading frame predicts a Slo protein of 1235 aa. Thus, a conserved N-terminal domain, containing almost all of the six probable transmembrane domains, is followed by a series of alternative yet related modules. This analysis predicts up to 144 different coding combinations. To determine which combinations could be readily detected in fly head mRNA, 102 Ml3 subclones derived from the PCR reaction were probed as dot blots using separate oligonucleotide probes for each of the alternative domains; 23 distinct combinations were cataloged. Furthermore, DNA sequence analysis of clones isolated from the fly head cDNA library provided 8 additional coding combinations. Neither analysis provided evidence for restricted combinations of alternative exons.
Functional Expression of Calcium-Activated Potassium Channels Membrane patches from oocytes that had been injected more than 3 days before with RNA transcribed from a s/o cDNA (Al/C2/E2/G5/10 unless otherwise stated; Figure 2) showed unitary currents of large amplitude when calcium was applied to the cytoplasmic side (Figure 3). The amplitude of the currents was linearly dependent on the holding potential and reversed at zero potential when the potassium concentration was equal (120 mM) on both sides of the membrane; the conductance was 123 it 6.6 pS (n = 6) (Figure 3). When the potassium concentration was changed on one or other side of the membrane, the unitary currents were well fitted by the constant field expression, I = P. F?. V. [[K’ln - [K+],exp[-VFiRqjlRT where F and R have their usual il - exp[-VFiRT]), meaning, [K’]” is the extracellular K’ concentration, and T is 293OK. For the three conditions (2.5 m M [K+],,, 120 m M [K+],; 120 m M [K’],, 120 m M [K’],; and 120 m M [K+],, 2.5 m M [K’],) the estimates for P were 2.6, 2.7, and 3.5 x IO-‘? cm3/s, respectively; the extrapolated zero current potentials were close to -lOO,O, and 100 mV, respectively, which are similar to the theoretical values for the potassium equilibrium potential. At a given [CaZ+],, the probability of opening of the channels was strongly voltage dependent. At 100 PM [Ca”], very brief openings occurred extremely rarely at -80 mV (Figure 3A); depolarization increased both
Clontng
and Expression
of gic,
211
Figure 2. The Central gion of Slo
/
:AACAGCCAAA
Re-
(A) Schematic depiction. Alternative forms of the same length (A, C, and E) are boxed. Alternative forms of variable length (C and I) are also indicated. Numbers refer to the number of amino acids in each region. (B) Amino acid sequence of Slo (315-1235). Sequences in the C region are presented with gaps to achieve alignment. Variants within the regions A, C, E, and C have been numbered from top to bottom. Underline indicates sequence with possible resemblance to calcium-binding domain.
\
/
and C-Terminal
S’ M Ax Ga ’ S,Ml
B
01-6 646
CKN
LATFRKGVRAVQMVGRAKDDEYSLSNEHHPAPTFTPPELPKRVHVRGSVSGDITRDRED’r I.ATFRKGVRAV;MVGRA.................................SDITRDR~DT
NL
I,T”QPRSKFDDLV............................................... I,TVQPRSKFDDL.............“~HHPAPTFTPPELPKRVHVRGSVSG”~TRDREDT I,T”QPRSKFDDI,......................................GDITRDREDT I)VGMTMMQTGMVNQGITSVMNTMF: DEHHPAPTFTPPELPKRVHVRGSVSGD~TRDR~~~T 111
LNRN’JR~I’N~~GNGTGGMHHMNNTAAAAAAAAAAAGKQVNKVK~jTV~SRQV~GQV~SPSQY~R
77 i
IO, 1 NlilDANPYAGYQLAYEVKKIIM
PTSKSSGTGTQNQNGG”SLPAGIADDOSKDFDFEKTEMKYDSTGk,b
847
HWSPAKSLEDCILI~RNQAAMTVl,NGHV\“V’CI,FnDPDSPI~GS
901
‘~I)YIRREirlKMLQNL.PKISVLNGSPLSHADLHRVNVT,.ADK~A~L,ASLN
916
I KIIM’~F’I,DTlG”I,SQHGPEFDNI.SATAOSFIVL~~DP
1043
DT~:I.‘/I.‘I’V~FACGT~FA”S’~~,DSL~ST’~YF~QNA~~,,IKSL1
1110 117
I’PE
I’GGA~I’CLCI.I1,REGAGLRGGYSTV
~:SI,SNK~~RCR\IGQTSI,~DPLA~~G~~~:K~G~~L~.'~A*LKSYG~L~IC:LYR~‘RL)I'SSSCDA~SKR~V~ /
'PN~'P"DPS~,~,PTD(,VFVL.M()FDPGLEYKPPAV~APAGC:RGTN',~GSGVGGGCSNKUDNS
the frequency of openings and the mean open time (Figure 3). Ensemble currents activated exponentially, becoming faster with stronger depolarization (Figure 4C). At 300 uM [Ca2+],, time constants were 3.9 + 0.31 ms, 3.4 & 0.30 ms, 2.7 k 0.17 ms, 2.6 + 0.12 ms, and 2.1 f 0.13 ms at +60 mV, +80 mV, +I00 mV, +I20 mV, and +I40 mV, respectively (n = 3-6). The current declined by less than 10% during depolarizations continued for 5-10 s (30 uM or 100 uM [Ca2+],, +80 mV, n = 5). Channel activity was absolutely dependent on [Ca2+],, even at very positive test potentials (+80 mV). At a given potential, the current increased with [Ca”], in the range l-100 PM (Figure 4), the most striking effect being to shift negatively the voltage at which the cu:rent activated (Figures 48 and 4D). The time
1235
course of activation of the current also became more rapid in higher [Caz’], (Figure 4C). At +80 mV, activation time constants were 27.0 + 1.2 ms, 13.8 + 1.6 ms, 4.0 f 0.2 ms, and 2.1 f 0.13 ms at 3 PM, IO PM, 100 PM, and 300 PM [Ca”],, respectively (n = 3-6). Many combinations of substituted blocks of amino acids in the A, C, E, G, and I domains are predicted from the isolated cDNAs. The functional properties of A1/C21E1/G3/10 were compared with those of Al/W E2/G5/10. There was no difference in the unitary conductance. However, at a given [CaZ’l, and potential, the channel openings of Al/C2/El/G3/10 were clearly much longer than those of Al/C2/E2/G5/10 (Figure 5). At 60 mVand 30 uM [Ca2+],, theopen timedistributions were fit by two exponentials (Figure 5). The time constant of the longer component was 97.1 f 16.1 ms
Neuron 212
Figure 3. Currents Channels
86 25 65 c4 E $3
80
s2 21 60 40-
OO 0
20M -20 “r”“m ”r”“““T””
10 20 Amplitude (PA)
30
c
-4oc -607
V W)
100lM 1
d---c
10vM
-
3pM
-
IIOPA
(A) Records of channel activity at different potentials (indicated beside each trace, mV). [Ca2’], was 100 PM, and potassium concentration on both sides of the membranepatchwas120mM.(B)Amplitudedistribution from a recording such as shown in (A) (at f60 mV). This is an all-points histogram (sampled at 1 kHz); the baseline noise is not shown (broken line). (0 Currentvoltage relation for unitary currents. Open circles: [K+], (bath) and [K+], (pipette) were both 120 mM. Closed circles: [K’], = 120 mM; [K’],, = 2.5 mM. Closed squares: [K’], = 2.5 mM; [K+],, = 120 mM. The mean current amplitude at each potential was determined from the amplitude distribution at that potential; points shown are means of these values from three to six patches, with SEM where this exceeded the size of the symbol.
Figure 4. Calcium tassium Currents
B
NPM
-50
s/o Potassium
potassium conductance in some tissues (Haylett and Jenkinson, 1990); neither affected s/o currents in oocytes (at +80 mV, 30 PM [Ca*+],, control: 9.0 + 1.0 nS, charybdotoxin [I uM]: 8.8 + 1.3 nS, n = 4; control: 3.4 f 0.24 nS, apamin [3 KM]: 3.1 f 0.27 nS, n = 3). On the other hand, tetraethylammonium (TEA) was a very potent channel blocker at the extracellular side (added to the pipette solution). At 100 uM TEA, openings of single channels were interrupted by many flickery closures; at 1 m M the unitary current traces were noisy and of reduced amplitude; and at IO m M unitary currents were not seen. TEA (100 PM) reduced
(n = 6) for Al/C2/El/G5/10 and 6.7 f 1.6 ms (n = 11) for Al/C2/E2/C5/10. Further systematic experiments are in progress to determine the contributions of the all the variable regions of the molecule to the gating kinetics and calcium sensitivity. A putative calcium-binding region is located within El (Figure 2B) (Atkinson et al., 1991); we expressed the channel in which these 12 residues were deleted by site-directed mutagenesis of Al/C2/El/G5/10. The prop erties of the expressed channel were not obviously different from those of the wild-type Al/C2/El/G5/10. Charybdotoxin and apamin blockcalcium-activated
A
through
50 0 Potential (mV)
C
Potential (InV)
100
Dependence
of s/o Po-
(A) Channel activity recorded at f40 mV In fourdifferentcalcium concentrationsfindicated by each trace). Each sample shown IS 500 ms. (B) Open probabilities at different [Ca’+], and potential. These observations were made on a patch containing only one active channel (no double openings were observed in 5 min of recording even at 300 uM [Cal],, +60 mV). A cursor set at 50% mean amplitudewas used to measureopen CT.,) and closed (1,) durations, and open probability was computed from s,/fs,, + T,). This illustration is from Al/C2/El/G3/10. (C) A membrane patch was depolarized from -60 mV to +80 mV for200 ms; records show averagecurrents recorded from 30such depolarizations. The depolarizing steps were repeated in fivecalcium concentrations (indicated beside each trace). (D) Voltage dependence of potassium conductance measured from ensemble records such as shown in (C). Calcium concentration indicated beside each trace; 3-11 patches for each point. In all records, potassium concentrations were 2.5 m M in the pipette and 120 m M in the bath.
Cloning 213
and Expression
of gkcr
B
A
Al/C2/El/GWlO
Ai/C2/EZ/G5/10
TEA was only a very weak blocker of the potassium current when applied to the cytoplasmic surface of the patch (10 m M inhibited by 24% + 2%, n = 8; 30 m M by 52.6% + 2.3%, n = 5; +I00 mV, 30 PM or 100 uM [Ca2’],). It might be thought that the low sensitivity to internal TEA correlates with the Ser residue at position 300. Other voltage-gated potassium channels have Thr in this position, but mutagenesis to Ser reduces IO-fold the affinity for internal TEA (Yellen et al., 1991). We expressed the mutant channels in which Ser-300 was replaced by Thr or by Ala; there was no change in the sensitivity to internal TEA. Discussion
D
C Al/C2/E2/G5/10
Al/C2/Ei/G3/10
120
120,
100
100 80 M) 40 i
Figure
5. Difference
between
Two s/o Variants
(A) and (C) are from Al/C2/E2/C5/10; (B) and (D) are from Al/C2/ El/C3/10. (A and B) Sample records at +60 mV in the calcium concentrations indicated. Al/C2/E2/C5/10 showed no openings when the calcium concentration was 10 PM. (C and D) Distribution of open times for two typical patches expressing Al/C2/E2/ C5/10 and Al/C2/El/C3/10, both at 60 mV in 30 uM [Ca’+],. The distributions are fitted by Alexp(-t/T,) + A,expt-t/r,); for Al/C21 E2/G5/10 A, = 202, 7, = 0.6 ms, A2 = 73, T> = 4.3 ms and for Al/ C2/El/G3/10 A, = 84, T, = 11 ms, Ar = 65, T> = 86 ms.
the ensemble current from 884 + 134 pA (n = 9) to 489 k 71 pA (n = 5; at +I00 mV); this high sensitivity to external TEA is characteristic of voltage-dependent, calcium-activated potassium channels of high unit conductance in many different cells (LaTorre et al., 1989; Haylett and Jenkinson, 1990; Adams et al., 1982; Yellen, 1984). The effectiveness of extracellular TEA is also consistent with the presence of a Tyr residue at position 308, because Tyr in the equivalent position of other voltage-dependent channels provides a high affinity TEA-binding site (MacKinnon and Yellen, 1990; Kavanaugh et al., 1991, 1992; Heginbotham and MacKinnon, 1992). Therefore, the s/o channel was expressed in which this Tyr was mutated to Val. Unlike the normal (wild type) channel, the mutant channel remained functional in the presence of IO m M external TEA (n = 15). This result indicates that, despite the differences in amino acid sequences between s/o and the Shaker-like channels, the same residues contribute to the external mouth of the pore (Guy and Conti, 1990; Durell and Guy, 1992).
The results identify the s/o gene product as a calciumactivated, voltage-dependent potassium channel. Although there is little primary sequence homology to other cloned potassium channels, several important residues in the hydrophobic regions are the same as those in Shaker-like channels, most notably the poreforming segment (Figure 1) (Wei et al., 1990; see Atkinson et al., 1991). Our assignment of membranespanning regions Sl and S2 differs from that suggested by Atkinson et al. (1991), to provide better alignment of several key residues in these regions (Figure 1; see also Durrell and Guy, 1992). Some of the conserved residues are also found in ion channels other than potassium channels; other amino acid residues, such as the Cys residues, which occur in S2 and S6 of all previously known potassium channels and the photoreceptor cation channel (Guy and Conti, 1990; Jan and Jan, 1990), are absent in Slo. The pore-forming region of the channel (usuallydefined as the 21 residue segment in the S5-S6 domain delineated by 2 Pro residues in Shaker, Shaw, Shab, and Shal; see Figure 1; Durell and Guy, 1992) is well conserved between s/o and other voltage-dependent channels in its C-terminal one-half, though not in the N-terminal one-half. Slo has a Cys residue in the position equivalent to the second of the 2 Pro residues; we found no obvious differences in the properties of s/o channels in which this Cys was changed to Pro by site-directed mutagenesis (data not shown). Immediately adjacent to this Cys is a Tyr residue in Slo (position 308; Figure 1). The rat brain potassium channel RBKI, which is a member of the Shaker family cloned from rat brain (Christie et al., 1989), has a Tyr residue (Tyr-379) in the analogous position. Indeed, all channels having a Tyr in this position are very sensitive to block by external TEA; the only substitution that retains the high affinity is Tyr to Phe, leading to suggestion that the aromatic electrons are important for the binding of TEA (Kavanaugh et al., 1991 and references therein; see also Heginbotham and MacKinnon, 1992; Kavanaugh et al., 1992). In RBKI, the replacement of Tyr by Val reduces the TEA affinity by about 30-fold. The present finding that the slo channel was very sensitive to TEA and that this sensitivity was dramatically reduced by the mutation Y308V, indicates that
NWKNl 214
the external mouth of the s/o channel has a threedimensional structure that is fundamentally similar to that of Shaker channels (Durell and Guy, 1992). The full significance of the abundance of alternatively spliced gene products from adult fly head cDNA remains unknown. The other families of potassium channels cloned from Drosophila, most notably the Shaker channels, rely on alternative splicing to generate structural diversity. Alternatively spliced Shaker variants have been shown to result in functional differences among the expressed channels (Schwarz et al., 1988; Timpe et al., 1988; Hardie et al., 1991), and we observed marked differences in the mean channel open time between variants Al/C2/E2&5/10 and Al/ C2/El/G3/10 (Figure 5). Finally, the different splice products that have been isolated all came from adult fly head cDNA; it is quite possible that they do not represent the full complement of mRNAs transcribed from this locus, because other developmental or tissue-specific forms may exist. The properties of the single-channel currents observed in oocytes expressing s/o resemble in several respects those of thecurrent that is missing in skeletal muscle fibers of the s/o Drosophila mutant (known as 7,8). The single-channel conductance is k, ICFI or &cd; somewhat higher (123 pS versus 88 pS) but the rapid activation by depolarization is similar. It has been deduced from electrophysiological studies in Drosophila muscles and neurons that current through s/o channels inactivates during a sustained depolarization; the currents in Xenopus oocytes did not inactivate very much during several seconds. Inactivation in Drosophila muscle might be secondary to a reduction in [Ca*+],, or Drosophila channels might form as heteropolymers with other subunits that confer distinct inactivation properties, as has been found for the Shaker family of channels (Schwarz et al., 1988). As in Drosophila muscle (Komatsu et al., 1990) and mammalian cells (Marty, 1981, 1989; Blatz and Magleby, 1987), channel activity persisted for hours in excised patches; this suggests that gating by calcium does not require other cytoplasmic proteins. There is a sequence in Slo (Figure 2) that is stated by Atkinson et al. (1991) to resemble the EF hand of calciumbinding proteins (see Marsden et al., 1990); its removal by site-directed mutagenesis made no obvious difference to the properties of the expressed channel. It will be important todetermine whether other putative EF-binding domains exist that are critical to calcium sensitivity. However, an important difference between the properties of the s/o channels in Xenopus oocytes and calcium-activated potassium currents in most other cells, including Drosophila muscle, is the tower sensitivity to [Ca?‘], in the oocytes. It is possible that a higher sensitivity to [Caz+], might be found for other splice variants that have not yet been expressed; it is also possible that these channels normally form as heteropolymers and that other, yet unknown, subunits contribute to the higher affinity for [Cal+],. s/o channels might also be insensitive to [Cai’], be-
cause they undergo posttranslational modifications in most cells, which do not occur in the oocyte. For example, the calcium sensitivity of potassium channels can be markedly altered by phosphorylation with A kinase (Ewald et al., 1985; Reinhart et al., 1991; Kume et al., 1989) or possibly by autophosphorylation (Chung et al., 1991). Slo has several amino acid sequences resembling those commonly phosphorylated by kinases, including a consensus sequence for A kinase (Ser-1013). The heterologous expression of this family of channels should make it possible to examine directly the role of such processes in the function of calcium-activated potassium channels. We anticipate that the cloning and expression of similar molecules will aid in clarifying the structural and functional relationships among the members of this protean channel family. Experimental
Procedures
Isolation of cDNAs Total RNA (0.4 mg) was extracted from 2 g of fly heads (Canton-5; courtesy of Dr. M. Forte) byguanidinium isothiocyanate and acid phenol extraction; 2 ug was reverse transcribed as described (Bond et al., 1991). Two sets of degenerate oligonucleotides, AK and B/D (synthesized on an Applied Biosystems 390A PCR Mate, Foster City, CA), were employed in nested PCR. The sequences were based on a predicted fragment of Slo amino acid sequence (Robertson et al., 1990, Sot. Neurosci., abstract). In the first reaction, the A and C chains (A = GCX(CT)TX(CA)CXTT(AG)ATGAC + CCX(CT)TX(CA)GXCTXATCAC; C = CC(AC)TAXCCXACAGT(AG)CTCAT + CC(AG)TAXCCXACXGT(AG)CTCAT) were used to amplify 5% of the reverse transcriptase reaction (IO cycles: 96’C for 30 s, 45OC for 30 s, 72°C for 30 s). One-tenth of this material was transferred to a fresh tube, and a new reaction was performed with B (CGACGAATTCCCXGA(TC)AT(ATC)TT(AG)CA (AC)TA + CGAGGAATTCCCXGA(TC)ATfATC)TToCA(AG)TA) and D tGCGCAACCTTA(AC)(AG)AAfAG)TAXACfAG)CAXGT) (40 cycles: 96OC for 30 s, 55OC for 30 s, 7Z°C for 30 s). All PCRs were performed using Replinase (NEN-DU Pont, Boston, MA) on a thermocycler (Model 50, Coy Laboratory Products, Grass Lake, MI). The resulting band was cut at the artificial restrictton sites introduced by the primers and subcloned into M 13 phage, and the nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using Sequenasr (US Biochemical, Cleveland, OH). Based on this DNA sequence, a unique oligonucleotide was synthesized (CCGGGCATTATACA TCTGCTGGAGAACTCTGGC), radiolabeled at the 5’ end with 74 polynurleotide kinase (BRL, Gaithersburg, MD), and used as hybridiration probe to screen 750,000 fly head cDNA clones rn kgtll (Papazian et al., 1987; provided by Dr. M. Forte) using Gene Screen membranes (NEN-DU Pant). DNA was prepared from plaque-pure phage; the inserts were subcloned in both onentations into Ml3 phage; and the nucleotide sequences were determined. The previously described s/o cDNA sequence did not include an initiator codon (Atkinson et al., 1991). The PCR was used to determine whether any of the 46 clones isolated from the fly head cDNA library contained sequences that predicted an extended N-terminal coding sequence. Pure plaques of each of the 46 clones were excised from agar plates, and phage DNA was eluted in IO ul ofwater; 1 ~1 was used in the PCR with chains that would amplify the known 5’ configuration. One chain was dtrected within the coding region (3’ chain; CAAAGCACCAC TACTTCCGCA), and the other was directed within the known, presumably introntc, sequence 5’ to the open readmg frame ti’ chain; CAACATTGGCGAGCCGCAGCG). Following amplification (40 cycles: 96OC for 30 s, 55OC for 30 s, 72OC for 30 $1, the reaction products were visualized on an ethidium bromide stained agarose gel. Under these conditions, 1 clone fhSP693-4)
Cloning 215
and Expression
of gKcr
did not amplify, suggesting that it contained adifferent sequence in this region. Analysis of s/o cDNA clones revealed alternative exon combinations in regions A, C, E, and G. This was studied in further detail by PCR on fly head cDNA. One primer (CATCCACCTCCATACGCTTGG) was directed to the N-terminal region between S4 and S5, and the other primer (G~G~CATGTCGTGCATGCC) was directed to an invariant part of the carboxyl terminus. Following 45 cycles (96OC for 30 s, 60°C for 30 s, 72OC for 30 S) the DNA was cut with Sphl and Sacl. Bands approximately 1350 bp in length were gel purified and cloned into Ml3 phage; nucleotide sequences were obtained as described above. This strategy was repeated twice with chains directed to other regions of the carboxyl terminus (5’: GTACGCCTGGCATGCACCAC; 3’: CCACGAATrCGTGCCGCAACTGCCACTCCTCGTC and 5’: GCACGAATrCAAGTGATATCGCCATCG; 3’: GCACAAGCTTGTACGAATAAGGACCATCAG). The transcription vector used for expression (pS-) was derived from pSelect (Promega, Madison, WI) by reversing the orientation of the fl replication origin. All in vitro transcription properties of pS are identical to those of pSelect. Two plasmids were used for oocyte expression (pSPexpAlC2E2G510 and pSPexpAlC2ElG310). Both contained the 900 bp Bglll-Sac1 fragment from 1SP693-4. encoding 170 bpof Suntranslated sequences, the initiator codon, and the first four likely transmembrane segments; pSPexpAlC2E2G.510 was constructed by ligating onto this a SaclKpnl fragment from one of the hybridizing h clones (hSP3-2) that contained the rest of the coding sequence and approximately 500 bp of 3’ untranslated sequence (in which resides a unique Sal1 restriction site). To construct pSPexpAlC2ElG310, a 1330 bp Sacl-Sphl fragment generated by PCR replaced the corresponding fragment from pSPexpAlC2E2G510. To ensure that PCR mistakes were not incorporated into plasmids used for expression studies, the nucleotide sequences for each region were determined to be identical to multiple representatives isolated from the fly head cDNA library. The complete nucleotide sequences of the coding regions for all expression plasmids were verified prior to RNA synthesis and injection. Expression plasmids were linearized at the Sal1 site, and capped mRNAs were syntheisized in vitro usingT7RNA polymerase. All molecular biology enzymes were purchased from BRL. Nucleotide and protein sequence computer analysis was performed using Genetics Computer Group software. Oocyte Expression Oocytes were injected with 50 ng of RNA in 50 nl of sterile water. Inside-out membrane patches were pulled from oocytes from which the vitelline membrane had been mechanically removed after brief (5-10 min) exposure to a hypertonic solution (220 m M aspartic acid, 220 m M N-ethylglucamine, 2 m M M&I,, 10 m M EGTA, 10 m M HEPES [pH 7.21). Except where otherwise stated, the pipette (extracellular) solution contained 2.5 m M potassium ,gluconate, 115 m M sodium gluconate, 1.8 m M calcium, and 10 m M HEPES (titrated to pH 7.2 with NaOH; final sodium concentration 119 mM), and the bath (intracellular) solution contained 116 m M potassium gluconate, 2 m M magnesium gluconate, and 10 m M HEPES (titrated to pH 7.2 with KOH; final potassium concentration 120 mM). Calcium gluconate was added in amounts expected to give the free calcium concentrations stated. These were computed from stability constants for calcium gluconate and magnesium gluconate (Dawson et al., 1969). Chloride-free !solutions were used to avoid currents through calcium-activated chloride channels endogenous to the oocyte. Electrode resislance prior to seal formation was about 2 MQ; seals weretypically ‘l-5 GQ. Calcium-dependent unitary currents were observed in oocytes from 3 to 30 days after injection with RNA, but the majority of experiments were carried out on oocytes that had been injected between 5 and 10 days previously. Single-channel currents were recorded with an Axopatch IB amplifier (front panel lilter 5 kHz) and played into an A/D converter (Axolab, Axon Instruments) at 1 kHz. Subsequent analysis was as described (Derkach et al., 1989; Shen et al., 1992). Numerical data are expressed as means ? SEM for the number of patches indicated.
Acknowledgments This work was supported by grants from the United States Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
March
31, 1992; revised
May 20, 1992
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to
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D., Jan, Y., and Jan, from ShakercDNA
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Accession
Number
The nucleotide sequences of all the cDNAs have been submitted to GenBank under accession number M96840.