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Department of Neurobiology and Behavior, Cornell University, Ithaca, New York
Submitted 6 March 2007; accepted in final form 2 April 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Robinson and Siegelbaum 2003; Santoro and Tibbs 1999). Ih is encoded by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel gene family. In vertebrates, there are four HCN genes (HCN 1–4), which give rise to channels that differ in their tissue distribution, channel properties and modulation by cyclic nucleotides (Ludwig et al. 1998; Mistrik et al. 2005; Notomi and Shigemoto 2004; Seifert et al. 1999; Stieber et al. 2003). cDNAs encoding Ih channels have been cloned from several invertebrates (Galindo et al. 2005; Gauss et al. 1998; Gisselmann et al. 2003, 2004, 2005a; Krieger et al. 1999; Marx et al. 1999;), including the spiny lobster Panulirus argus (Gisselmann et al. 2005b). With the exception of the sea urchin (Galindo et al. 2005), there appears to be only a single gene for Ih channels in these invertebrates, but there is significant alternative splicing of its mRNA to generate functionally distinct channel subtypes (Gisselmann et al. 2004, 2005a).
We are studying the role of Ih in the pyloric central pattern generator network in the stomatogastric ganglion (STG) of the California spiny lobster, Panulirus interruptus. This 14-neuron network contains six major cell types with unique electrophysiological properties (Harris-Warrick et al. 1992). Although Ih is present in all six pyloric cell types, it differs substantially among them in its amplitude, voltage dependence of activation, kinetics, and sensitivity to neuromodulators (Peck et al. 2006). Several mechanisms could underlie pyloric Ih diversity, including differential gene expression, alternate splicing, and posttranslational modification. Alternative splicing is an important mechanism for generating structural and functional diversity of many membrane proteins, including ion channels. Although alternative splicing has already been reported in honey bee and fruit fly Ih channel genes (Gisselmann et al. 2004, 2005a), little is know about alternative splicing of the lobster Ih gene and whether this affects lobster Ih channel properties. Thus our first goal of this study was to look for alternative splicing in the P. interruptus Ih gene.
We previously showed that Ih interacts with IA in shaping postinhibitory rebound in the lateral pyloric (LP) cell (Harris-Warrick et al. 1995). Artificially increasing IA amplitude by microinjecting shal RNA into pyloric neurons provoked an endogenous compensatory increase of Ih, leaving the electrophysiological properties of the injected neurons unchanged (MacLean et al. 2003, 2005). To investigate whether this compensatory response is bidirectional, Zhang et al. (2003) overexpressed PAIH, the Ih gene from the related species P. argus in PD neurons; they did not detect any compensatory enhancement of IA. Instead the increased Ih significantly changed the firing properties of the PAIH-injected PD neurons (Zhang et al. 2003). However, it is possible that the native gene from P. interruptus might have some different sequence that would evoke an upregulation of IA.
To address these questions, we cloned PIIH, the Ih gene from P. interruptus. We found that this gene has multiple alternative exons, generating 
10 different transcripts. When expressed in Xenopus oocytes, each splice variant expresses different PIIH channel properties. Overexpression of two splice variants in pyloric neurons evokes an increase of Ih that is not accompanied by a compensatory increase in IA. PIIH overexpression enhances the firing properties of pyloric neurons in an amplitude-dependent manner.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Total RNA was isolated from lobster nervous system (brain, abdominal, and thoracic ganglia) using the RNAqueous-4PCR kit (Ambion, Austin, TX). DNA contamination in the total RNA was removed by treatment with DNase I (Ambion, Austin, TX) at 37°C for 30 min.
5' rapid amplification of cDNA ends (5' RACE)
The gene-specific primers GSP1 (5'-AAAGGCGGAGGAGGCGAACCAGGGAGAG-3') and NGSP1 (5'-AATATCGTGTCGGAGAGGCAGTTGAAGG-3') were designed based on the gene sequence of PAIH (GenBank: AY280847 [GenBank] ) and the sequence of a PIIH partial clone (kindly provided by Dr. G. Gisselmann). Cloning of the 5' ends of the cDNA was performed using the 5'RACE Systems for rapid amplification of cDNA ends kit (Gibco-BRL, UK) according to the supplier's instruction. The RACE products were subcloned into pGEM-T Easy Vector (Promega, Madison, WI) and sequenced.
Reverse transcription-PCR (RT-PCR)
The full-length open reading frame of PIIH was amplified by RT-PCR in two independent reactions, using two different reverse transcriptases, SuperScript III RT (Invitrogen, Carlsbad, CA), and BD PowerScript RT (BD Biosciences Clontech, Palo Alto, CA), and two different and superior proof-reading DNA polymerases, platinum Pfx DNA polymerase (Invitrogen) and ProofStart DNA Polymerase (Qiagen, Valencia, CA). Five micrograms of total RNA were reverse-transcribed into the first cDNA strand using primer oligo dT. The first strand of cDNA was used for the first amplification with the primer pairs PIihD (5'-CGGGCACTCAAAGACGACATCAAGA-3')/oligodT2 (5'-AAGCT27VN-3'). A nested PCR was performed using 1 µl of the first amplification products with primers PIihA (5'-CCCGAGATGAATTACCGCGATGTGAG-3') and PIihB (5'-GGGCGCCCTGCTATTGGAACGAG-3'). The nested PCR products were subcloned into the pDrive cloning vector (Qiagen) and verified by sequencing.
Xenopus oocyte expression
PIIH splice variant DNAs were linearized with Mlu I and transcribed in vitro using a SP6 Message Machine kit (Ambion). The capped transcripts were cleaned using the RNeasy mini kit (Qiagen). Stage V to VI oocytes were surgically removed from female frogs during anesthesia in 0.15% MS222 (3-aminobenzoic acid ethyl ester). The eggs were then treated with 1 mg/ml collagenase type IA in solution containing (in mM): 82.5 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.5) for 60 min. The oocytes were defolliculated, but the vitelline membrane was not removed. Isolated oocytes were injected with 40 nl of PIIH cRNAs (250–500 ng/µl) and cultured in ND96 solution containing (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2(·2H2O), 1 MgCl2, and 5 HEPES (PH7.6) supplemented with 50 mg/l gentamicin, 2.5 mM Na pyruvate, and 5% horse serum for 3–4 days until recording.
STG dissection and PD cell identification
Adult California spiny lobsters, P. interruptus, were obtained from Don Tomlinson Commercial Fishing (San Diego, CA) and maintained in artificial sea water at 16°C until use. Lobsters were anesthetized by keeping them on ice for 30 min before dissection. The STG, along with its motor nerves and associated commissural and esophageal ganglia, was dissected and pinned in a silicone elastomer (Sylgard)-coated dish containing saline as described by Mulloney and Selverston (1974). The physiological saline solution consisted of (in mM): 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10.0 MgSO4, 2 glucose, and 11.1 Tris base, pH 7.4 (Mulloney and Selverston 1974). The PD neurons were identified during intracellular recordings (3 M KCl, 10–25 M
) by their typical membrane potential oscillation shapes and synaptic inputs (Kloppenburg et al. 1999).
RNA microinjection into neurons
After neuronal identification, PD neurons were injected with RNA using pressure pulses (40 psi; 0.2 Hz, 30- to 70-ms duration) driven by a homemade pressure injector and a pulse generator (Master-8; AMPI, Jerusalem, Israel). The RNA solution contained 0.25–0.5 µg/µl PIIH or GFP(control) cRNA and 0.08% Fast Green to monitor the injection. After injection, the whole preparation was incubated in sterilized recording saline without Tris base but containing 5 mM HEPES, pH7.4, 2 g/l glucose, 100,000 unit/l penicillin, and 100 mg/l streptomycin at 16°C for 4–5 days to allow protein expression.
Electrophysiology
XENOPUS OOCYTES. A standard two-microelectrode voltage clamp was used to measure the current properties of the PIIH splicing variants. The oocytes were voltage clamped using a Geneclamp amplifier driven by Clampex 8.0 software (Axon Instruments). All recording were made in standard ND96 solutions without gentamicin, Na pyruvate, and horse serum. For some experiments, 10 mM Cs+ or 100 µM ZD7288 were included.
Microelectrodes were filled with 3 M KCl and had a tip resistance of 1–5 M. To measure Ih, the cells were held at –40 mV, and the voltage dependence of activation was measured with a series of 8-s hyperpolarizing voltage steps in 5-mV increments from –50 to –120 mV at 20-s intervals. These steps were not leak subtracted, so an instantaneous leak current is detectable at the beginning of the step; this value was subtracted from the amplitude of Ih (see following text). The reversal potential of Ih was measured from the tail currents after a preactivating pulse to –100 mV for 8 s with a series of 4-s pulses from –70 to +30 mV in 10-mV increments. These protocols were used in all of the PIIH splice variants except PIIH-I. For PIIH-I, voltage activation curve was measured with hyperpolarizing voltage steps from –70 to –140 mV, and the preactivating pulse for measuring the reversal potential was to –120 mV.
The effect of cAMP was tested by recording the basic parameters before and after switching to a bath solution containing the membrane-permeable cAMP analog, 8-Br-cAMP. Perhaps due to the presence of the vitelline membrane on the oocytes, bath application of 1 mM 8-Br-cAMP caused only a subtle modulation of the activation kinetics and voltage-dependent activation of PIIH channels after 1.5 h. To accelerate the rate of increase in intracellular cAMP in the large oocytes surrounded by a vitelline membrane, we increased the concentration of 8-Br-cAMP to 10 mM; responses to this larger dose were seen within 30 min.
PD NEURONS.
After 4–5 days in organ culture, PD neurons were voltage clamped using an Axoclamp 2A amplifier and pClamp8 software (Axon Instruments, Foster City, CA). Microelectrodes were filled with 3 M KCl and had a tip resistance of 8–10 M for voltage recording and 
8 M for current injection. To isolate PD neurons from most synaptic input and to isolate Ih and IA from most other currents, we superfused the ganglion with saline containing 10–7 M tetrodotoxin, 5 x 10–6 M picrotoxin and 20 mM tetraethylammonium chloride (TEA). For some experiments, 5 mM CsCl or 100 µM ZD7288 were added. Ih was recorded using similar protocols as in oocytes, except that the voltage dependence of activation was measured with 8-s hyperpolarizing voltage steps from –45 to –120 mV. Because the time constant of activation is slow, we did not use leak subtraction for measuring Ih. To measure IA, the cells were held at –50 mV and the voltage dependence of activation was measured following a deinactivating prepulse to –120 mV for 400 ms and then a series of 400-ms voltage steps from –50 mV to +40 mV in 10-mV increments. A control protocol for activation of non-IA currents was the same as the activation but without the deinactivating step to –120 mV. Traces were leak subtracted using a P/6 protocol with steps opposite to the sign of activation. The control protocol currents were digitally subtracted from the activation protocol currents to isolate IA.
Current analysis
Ih amplitudes were measured from single exponential fits of the data performed in Clampfit, version 9.0 (Axon Instruments), extrapolated back to the beginning of the hyperpolarizing step (at the point of the leak current) and forward to approximate the steady state at 10 s. Currents were converted to conductances, using a reversal potential (Vrev) of –40 mV for oocytes and –30 mV for PD neurons (Zhang et al. 2003). The conductance-voltage data were fit to a first order Boltzmann equation
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). The resulting g/V curve was fitted to the Boltzmann relation (Eq. 1) but with n = 3. Analysis of rhythmic activity
We analyzed rhythmic activity in PD neurons using Spike2 (Cambridge Electronic Design, Cambridge, UK). The minimal membrane potential (Vmin) was measured at the most hyperpolarized potential in the trough of the oscillation. The oscillation amplitude was the difference between Vmin and the most depolarized potential of the slow wave oscillation (at the base of the action potentials, Vmax). The time to the first spike was the time from Vmin to the top of the first spike. The cycle duration was the time between the Vmin of two adjacent oscillations, and the duty cycle was the burst duration divided by the cycle duration. All measures were based on average measures of 40 cycles.
Statistics
All values are given as the mean ± SD. Statistical significances were determined using ANOVA and Student's t-test. Regression lines were plotted, and R values determined using SigmaPlot 10.0 (Systat Software).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Our cloning of the Ih gene from P. interruptus began with a partial transcript of PIIH generated by Dr. Günter Gisselmann (Ruhr-University Bochum, Germany). This clone starts from the second transmembrane (TM) domain and extends to the carboxy-terminus of the protein. The sequence information and the gene sequence of PAIH, the homologous gene from P. argus (Gisselmann et al. 2005b) were used to design reverse primers GSP1 and NGSP1 to amplify the 5' end of PIIH cDNA by 5'RACE. A 550-bp fragment was obtained, and sequence analysis revealed that the cDNA fragment shares high degree of sequence similarity to the 5' terminus of PAIH. The deduced amino acid sequence contains the first and second TM domains and the entire amino-terminus beyond the start methionine, which was identified by a contiguous consensus Kozak sequence. Therefore we considered the 550-bp fragment as the 5' end of PIIH cDNA.
To isolate possible full-length splice variants of the PIIH gene, RT-PCR was performed to amplify the full open reading frame of PIIH. Two independent reactions were carried out using different reverse transcriptases and separate proof-reading DNA polymerases (see METHODS for details) to confirm the presence of splice variants. In all, 88 independent clones with insert were isolated, and 33 of them were completely sequenced. This revealed 10 different splice variants of PIIH, with lengths varying from 2,025 to 2,157 bp, coding for proteins of 674–718 amino acid residues and calculated molecular mass of 76.7–82.1 kDa. Each of these 10 splice variants was independently isolated in both of the independent RT-PCR reactions. Although these are probably the most frequently expressed variants, it is certainly possible that additional splice forms exist that did not show up in our screen.
From their amino acid sequences, we found that the PIIH gene shows alternative splicing at five separate points in its open reading frame (Fig. 1A) : in the S3–S4 linker and the S4–S5 linker sequences, in the pore-forming P-loop and entire S6 transmembrane domain, in the cyclic nucleotide binding domain (CNBD), and near the 3' end of the coding sequence. First, 7 of the 10 splice variants contain a 15-bp-length cDNA insert in the interloop region between the S3 and S4 TM domains, coding for 5 additional amino acids (segment A; Fig. 1B). This insert clusters four charged amino acids (2 positively charged amino acids, 2 negative charged amino acids), adding significant charge to this region of the protein. The remaining three variants lack the additional insert at this site. Second, 5 of the 10 variants have an additional 9-bp sequence coding for three amino acids (1 hydrophobic, 2 hydrophilic; segment BS) in the S4–S5 interloop region; 2 of the 10 variants have a second alternative long insert of 117 bp coding for 39 amino acids (segment BL) at the same site (Fig. 1B). The remaining three variants have no additional insert at this site. Third, there are two alternative pore-forming regions, beginning 5' to the P-loop and extending shortly past the S6 transmembrane domain, in the PIIH gene (Fig. 1B). Pore I is found in seven of the splice variants and pore II in the other three variants. These two alternate segments are the same length and are very similar to each other, with only 10 differences in 73 amino acids (Fig. 1B). Seven of these differences are conservative changes. However, the first amino acid of this segment is an uncharged T in pore I but a negatively charged D in pore II; the third amino acid of this segment is a hydrophilic H in pore I, but a hydrophobic F in pore II; the 26th amino acid, immediately after the GYG triplet, is either an uncharged S in pore I or a positively charged R in pore II (Fig. 1B). Thus these pore segments differ by two charges. Fourth, in the C-linker region beyond the S6 TM domain and the 5' end of the cyclic nucleotide binding domain (CNBD), there are two alternative segments of 34 amino acids. Segment C2 is identical to the corresponding sequence in PAIH (Gisselmann et al. 2005b); it is only found in one variant, and differs from segment C1 in all the other variants by nine amino acids (Fig. 1B). Four of these differences are conservative changes. In the other five changes, two hydrophobic amino acids (F, A) in segment C2 are replaced by two hydrophilic amino acids (Y, S) in segment C1; two hydrophilic amino acids (Y, Q) in segment C2 are replaced by two hydrophobic amino acids (F, L) in segment C1; and a positively charged amino acid (R) in segment C2 is replaced by a hydrophobic amino acid (G) in segment C1. Thus segment C2 has one net positive charge compared with segment C1. Fifth, near the end of the carboxyl-terminus, there are two alternate segments. Segment D2 with nine amino acids is found in one variant, whereas all the other splice variants have segment D1 (8 amino acids; Fig. 1B); segment D2 is somewhat more hydrophobic than segment D1.
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As indicated in Fig. 1B, PIIH channels are subject to posttranslational modifications. The PIIH protein is predicted to have nine potential phosphorylation sites for protein kinase C (* in Fig. 1B; 1 is in alternate segment BL), eight putative phosphorylation sites for casein kinase (^ in Fig. 1B; 1 is in alternate segment D1), three putative sites for tyrosine kinase phosphorylation (# in Fig. 1B), and one putative protein kinase A phosphorylation site (the dot found in segment BL).
The protein sequence (represented by PIIH-I) shows a high degree of evolutionary similarity with previously cloned Ih channels from invertebrates and vertebrates: 95.7% identity with PAIH from the spiny lobster, P. argus; 82.9% identity with AMIH from the honeybee A. mellifera; 70.7% identity with the Drosophila homologue, DMIH; 49.6% identity with SPIH from the sea urchin S. purpuratus and 47.4% identity with mouse HCN1 (Fig. 1C). Overall the sequence similarity is particularly pronounced in the transmembrane domains S1–S6, the pore region, and the cyclic nucleotide binding domain, whereas the N- and C-termini and the interloop regions are less well conserved. The most similar PIIH variant to PAIH homologue from P. argus is ABSC2-I (96.4% identity). They differ by only 27 amino acids which are located in the S3–S4 linker and close to the end of the C-terminus.
Electrophysiological characterization of PIIH splice variants
Alternative splicing to generate multiple proteins from a single gene sequence is found in many ion channels (for example, Baro et al. 2001; Kim et al. 1997), including Ih channels (Gisselmann et al. 2004, 2005a). Changes in alternate exon expression can affect the voltage dependence of Ih channel gating, the activation or deactivation kinetics of the channel, and its response to cyclic nucleotide modulation (Gisselmann et al. 2003, 2004, 2005a). We have previously found that the biophysical parameters of Ih vary among the different cell types of the pyloric network: although all six pyloric cell types have Ih, they differ in the amplitude, Vact, and slope parameters of Ih (Peck et al. 2006). Could alternative splicing change the biophysical properties of PIIH channels? We addressed this question by heterologously expressing, in Xenopus oocytes, 9 of our 10 splice variants (except PIIH ABS -II, which was cloned into a sequencing vector and could not be used directly for in vitro transcription due to an additional upstream ATG in the vector). Careful comparisons of variants which differ by only a single segment allowed us to explore the roles of these segments in shaping the properties of the resulting currents.
Functional expression of alternate PIIH pore region variants in Xenopus oocytes
The sequences of variant PIIH A-I and PIIH A-II are identical except for the pore-forming region. PIIH A-I contains pore I, whereas PIIH A-II contains pore II (Fig. 2A). As stated in the preceding text, pores I and II differ in 10 of 73 amino acids with changes in charges at two sites (Fig. 1B).
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< 0.01. Although the PIIH A-I and PIIH A-II variants differed in their voltage dependence of activation and activation kinetics, they had similar ion permeability. The reversal potential Vrev measured from the amplitudes of tail currents after steps to varying voltages from an activating prestep to –100 mV (Fig. 2D) was around –40 mV (Fig. 2E) in 2 mM extracellular K+ for both channels. In addition, both currents produced by PIIH A-I and PIIH A-II were rapidly blocked by 10 mM extracellular Cs+ (Fig. 2F) or the specific Ih blocker ZD7288 (100 µM; data not shown). These data indicate that alternative splicing in the pore-forming region of PIIH can result in markedly different current properties but does not change the channel's ion permeability. The pore II variant has much faster activation kinetics and a more positive voltage range for activation than the pore I variant.
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There are six PIIH splice variants with alternate segments in the S3–S4 or S4–S5 interloop regions: PIIH-I, A-I, BS-I, ABS-I, ABL-I, and ABL-II. They differ only by the presence or absence of segments A, BS, and BL in the S3–S4 and S4–S5 interloop region (Fig. 3A). All these variants contain pore I except variant PIIH ABL-II (Fig. 3A). Previous studies of Kv and HCN channels have demonstrated that the S3–S4 linker influences activation gating (Gonzalez et al. 2000, 2001; Lesso and Li 2003; Tsang et al. 2004a, 2004b; Henrikson et al. 2003). In addition, the S4–S5 linker couples voltage sensing and activation of channels (Chen et al. 2001; Decher et al. 2004; Gisselmann et al. 2004). We thus expected that these inserts would change the activation phenotype of the channels.
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ALTERNATE SPLICING IN THE S3–S4 INTERLOOP REGION.
PIIH-I has no insert in any interloop region. PIIH A-I is identical to PIIH-I except that it has segment A in its S3–S4 interloop region, which adds a cluster of two positively charged and two negatively charged amino acids (Fig. 1B). The current produced by PIIH-I had the most hyperpolarized V1/2 (–107 ± 2 mV, n = 15) and the slowest activation kinetics (
m 4,600 ± 1,100 ms at –120 mV) of any of the six variants. We had to hyperpolarize the PIIH-I-expressing oocytes to more negative voltages (–140 mV) to obtain good Boltzmann fits of the steady-state currents, due to the very negative voltage activation curve for this variant. PIIH A-I, containing the additional cluster of charged amino acids in the S3–S4 linker, activated almost threefold more rapidly than PIIH-I (Table 1). In addition, the V1/2 was shifted by 
9 mV in the depolarizing direction (Fig. 3C). These changes were highly significant (P < 0.05). Thus this cluster of charged amino acids facilitates opening of the channel.
ALTERNATE SPLICING IN THE S4–S5 INTERLOOP REGION.
This region has three alternatives: no exon, segment BS, or segment BL. PIIH-I (with no exon in this region) could be compared with PIIH BS-I, which is identical except for a hydrophilic insert of three amino acids (segment BS) in the S4–S5 linker (Figs. 1B and 3A). As with PIIH A-I, insertion of segment BS accelerated the time constant for activation at –120 mV (from 4,600 ms in PIIH-I to 1,800 ms in PIIH BS-I); this insert also depolarized the voltage for half-maximal activation by 18 mV to –89 ± 4 mV (n = 22; P < 0.05 for both comparisons with PIIH-I; Fig. 3C, Table 1). The 39 amino acid segment BL is found in PIIH ABL-I; thus it can be compared with PIIH A-I. PIIH ABL-I did not express a current in oocytes (n = 11). To verify that this was due to BL alone, we also expressed PIIH ABL-II, which is identical to PIIH ABL-I but contains pore II instead: this also did not express in oocytes (n = 17). A search of the BL sequence showed that it contains a typical ER-retention/retrieval motif (RKR; Fig. 1B), which could be the reason that we could not detect currents with these isoforms (see DISCUSSION).
ADDITIVE EFFECTS OF INSERTIONS IN S3–S4 AND S4–S5 INTERLOOP REGIONS.
PIIH ABS-I contains both segments A (in the S3–S4 interloop region) and BS (in the S4–S5 interloop region), allowing us to study the interactions of these segments. We found that the effects of segments A and BS on current properties were additive. The current generated by PIIH ABS-I had even faster activation kinetics (m of 800 ± 190 ms at –120 mV) than PIIH A-I or PIIH BS-I. In addition, it was activated at significantly more depolarized potentials (V1/2 of –82 ± 3 mV, n = 30) than PIIH A-I and PIIH BS-I (P < 0.01 for all comparisons; Fig. 3C, Table 1).
Functional expression of PIIH carboxy-terminal variants in Xenopus oocytes
We next measured the effects of alternative splicing at the end of the C-linker and beginning of the CNBD by comparing the currents generated by PIIH ABSC2-I, which contains segment C2, with that by PIIH ABS-I, which contains segment C1. The current traces and voltage conductance curves for these variants are shown in Fig. 4. C1 and C2 differ by only nine amino acids (Figs. 1B and 4A), which change the hydrophobicity at several sites and add a positively charged amino acid (R) to segment C2. Similarly, we measured the effects of alternative exons near the C terminus by comparing the currents generated by variants PIIH BSD2-I and PIIH BS-I: these are identical except for the presence of segments D2 or D1 near the C-terminus. PIIH BSD2-I is more hydrophobic near the end of the carboxyl-terminus. Unlike the other alternative splice domains, alternative splicing in these two regions did not significantly change the kinetics or the V1/2 of the currents. The only significant change was in the slope of activation. The slope factor was larger for the variant PIIH ABSC2-I (segment C2 in the CNBD) than for PIIH ABS-I (segment C1) but very slightly smaller for the C-terminal variant PIIH BSD2-I (segment D2) than PIIH BS-I (segment D1; Table 1, Fig. 4C). Note that the effect of adding segment A noted in the preceding text is also seen when comparing the depolarizing shift in V1/2 and acceleration of activation in PIIH ABS-I when compared with PIIH BS-I (Fig. 4, B and C).
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Because PIIH splicing variants PIIH-I, A-II, A-I, BS-I, ABS-I, BSD2-I, and ABSC2-I all generated distinct hyperpolarization-activated cation currents that differ in their kinetics and voltage dependence of activation, we asked whether they differ in their extent of modulation by cAMP. For these experiments, we sought conditions in which a single oocytes could serve as its own pre-cAMP control (see METHODS). Bath application of 10 mM 8-Br-cAMP shifted the activation curves of all the PIIH splice variants in the depolarizing direction and significantly accelerated their activation kinetics (Fig. 5A). The largest effects of cAMP on the voltage dependence of activation were on PIIH ABSC2-I (the only variant with segment C2 in the CNBD) and PIIH A-II (the only expressing variant with pore II): the currents' V1/2 was shifted in the depolarizing direction by 16 and 14 mV, respectively (Fig. 5, B and C). The remaining variants were also modulated by cAMP, albeit to a lesser extent; the depolarizing shifts of the activation curves varied between 2 and 11 mV (Fig. 5, B and C). PIIH-I, with no optional segments, was the least modulated by cAMP compared with other variants (P < 0.05); the V1/2 was only shifted by 2 mV, although this was still significant (P < 0.05) compared with the unmodulated state (Fig. 5, B and C). As can be seen in Fig. 5C, the alternate pore II in PIIH A-II significantly enhanced its sensitivity to cAMP when compared with pore I-containing PIIH A-I (P < 0.01). Similarly, at the end of the C-linker and beginning of the CNBD, alternate segment C2 in PIIH ABSC2-I enhanced cAMP sensitivity when compared with the C1 variant PIIH ABS-I (P < 0.05). Thus splicing in the pore-forming region and the CNBD significantly altered the sensitivity to cAMP modulation of the channel.
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Overexpression of PIIH ABS-I in PD neurons produces an increased H-current but doesn't affect the expression of IA
We previously showed that overexpression of PAIH, the Ih gene from P. argus failed to evoke a compensatory increase in IA or other outward currents when injected into PD neurons and consequently modified their firing activity (Zhang et al. 2003). However, this was not the endogenous gene for our species, PIIH. There are significant amino acid sequence differences between our PIIH splice variants and the single PAIH sequence so far reported (Gisselmann et al. 2005b): PAIH does not contain the S3–S4 segment A, the S4–S5 segment BL, the C-linker and CNBD segment C1, the C-terminal segment D2, or the pore II segment; it also has additional differences in 20 amino acids toward the end of the C terminus. To further explore this question and understand how Ih contributes to pyloric motor pattern generation, we injected cRNAs from PIIH ABS-I or PIIH A-II into PD neurons and measured how this altered the neurons' electrophysiological properties. We chose these two variants because they contain most of amino acid sequences that are not found in PAIH (e.g., segment A, C1 and pore II. Note that BL could not be tested as channels containing this exon did not express); they cover all the C-terminal amino acid differences; and their currents were activated quickly at significantly more positive potentials when they were expressed in Xenopus oocytes. We took advantage of the fact that there are two PD neurons in each STG with very similar baseline electrophysiological properties. Thus one PD was injected with PIIH ABS-I or PIIH A-II RNA, whereas the other was injected with GFP RNA as a control; we previously showed that GFP expression does not change the firing properties of PD neurons (MacLean et al. 2003). Because we obtained qualitatively similar results from both variants, only PIIH ABS-I data are shown here.
Our results with PIIH ABS-I were similar to those previously found with PAIH RNA injections (Zhang et al. 2003). Hyperpolarizing steps from –40 mV to more negative values (–45 to –120 mV) produced a much larger Ih in PIIH ABS-I-overexpressing PD cells than in GFP-expressing PD cells (Fig. 6A) : the average maximal conductance was increased nearly threefold (n = 16 paired PDs, Table 2). Consistent with the rather depolarized V1/2 of PIIH ABS-I in oocytes, the current in the PIIH ABS-I-expressing PD neuron was activated at significantly more depolarized potentials than control PD neurons: the activation curve shifted 9 mV to more depolarized voltages (–81 ± 4 vs. –90 ± 6 mV), whereas the slope factor decreased by 3.8 mV. Furthermore, the current had a time constant of activation that was significantly faster than the control. For example, at –100 mV, Act was 2.2 ± 0.5 s for PIIH ABS-I-expressing PD neurons and 3.4 ± 0.8 s for the controls (Table 2, Fig. 6, A and B). The Ih in PIIH ABS-I-expressing PD cells was blocked by 5 mM Cs+ or 100 µM ZD7288, indicating that the increased inward current is a typical H-current (Fig. 6E).
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PIIH ABS-I overexpression alters the firing properties of the PD neurons in an amplitude-dependent manner
In the presence of modulatory inputs from other ganglia, the PD neurons in the pyloric network oscillate and fire rhythmic bursts of action potentials with a characteristic oscillation amplitude, number of spikes per burst, duty cycle, and phasing relative to other pyloric neurons. These parameters of activity might vary with large changes in Ih. When Zhang et al. (2003) overexpressed the homologous PAIH from P. argus in a PD neuron, its firing properties changed, compared with the control, Fast Green-injected PD neuron in the same ganglion. Increased Ih depolarized the minimum membrane potential of the cell, reduced the oscillation amplitude, decreased the time to the first spike, and increased the duty cycle and number of action potentials per burst. We tested whether overexpression of PIIH ABS-I would cause similar changes in PD neurons when compared with paired GFP-injected controls from the same ganglion.
Seven of 13 paired PD neurons displayed little change in firing properties after PIIH expression. On the other hand, the remaining six paired PD neurons did show significant changes in firing properties (Fig. 7A). In these preparations, the PIIH ABS-I-expressing PD fired more action potentials per burst (9 ± 2 vs. 7 ± 1) with longer burst duration (0.21 ± 0.07 vs. 0.18 ± 0.05 s), increased duty cycle (0.30 ± 0.09 vs. 0.25 ± 0.08), had more depolarized Vmin (-56 ± 3 vs. –60 ± 3 mV) and Vmax (–40 ± 5 vs. –44 ± 6 mV), and showed a decreased time from the oscillation minimum to the first spike (0.35 ± 0.1 vs. 0.38 ± 0.1s; P < 0.05, for all comparisons).
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0.7), but only the relationships between slope factor and the changes in firing properties (the number of spikes/burst, burst duration and the duty cycle) were statistically significant (P < 0.05). These results show that an increase in PIIH changes the firing properties of PD neurons; however, the change had to be large, with a threshold of two- to threefold increase in Ih before the neuron changed its activity pattern. |
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As previously seen with other invertebrate HCN genes, PIIH shows extensive alternative splicing of its RNA, generating 10 different transcripts. Most of the alternative segments change the properties of the resulting ion channels.
Alternative splicing in the pore-forming region has not been reported in other Ih channels (Gauss et al. 1998; Gisselmann et al. 2003, 2005a; Ludwig et al. 1998; Marx et al. 1999), but we previously reported two alternate pore domains in lobster shaker potassium channels (Kim et al. 1997). The alternate pore I and pore II segments of PIIH differed by only 10 amino acids; 3 of these changed either charge or polarity (Fig. 1B), but this resulted in a significant change in the channel properties. The Pore II variant had much faster activation kinetics and a more positive voltage range of activation, and was more sensitive to cAMP modulation than the pore I variant (Figs. 2 and 5C), but the two pores had the same reversal potential. The residue immediately after the GYG ion permeation motif was either a polar serine (pore I) or a positively charged arginine (pore II). This site is variable in other Ih channels (Gauss et al. 1998; Gisselmann et al. 2003,2005a; Ludwig et al. 1998; Marx et al. 1999), and mutating the charge at this residue in HCN1 alters voltage activation and gating kinetics without changing the ion selectivity of the channels (Azene et al. 2003). Site-directed mutations of other residues in the pore-forming region gave similar results (Azene et al. 2005; Xue and Li 2002).
Splicing in the interloop regions and PIIH channel activation
Previous studies have demonstrated that the extracellular S3–S4 linker is a determinant of activation in various K+ and Ca+ channels (Gonzalez et al. 2000, 2001; Nakai et al. 1994). We found a similar role in PIIH as seen by the effects of adding segment A, seen in our comparison of variants PIIH-I and PIIH A-I or PIIH BS-I and PIIH ABS-I. Segment A adds 5 amino acids and a cluster of 4 charged amino acids in the S3–S4 linker. Channels with segment A showed more rapid activation kinetics and more positive voltage dependence of activation than those lacking segment A. Mutational studies on HCN1 channels indicate that both the length and the amino acid constituents of the S3-S4 linker influence gating in similar ways to our results (Henrikson et al. 2003; Lesso and Li 2003; Tsang et al. 2004a,b). Increasing the length and introducing a cluster of charged amino acids in the S3–S4 linker may alter the energy barriers for channel opening, and influence gating by shaping the transmembrane electric field surrounding the N-terminal portion of S4.
The S4–S5 linker is joins the intracellular end of the S4 voltage sensor domain to the S5-S6 pore-forming domains, making it a candidate for the structural link between the voltage sensor and the activation gate. Research on HERG K+ (Tristani-Rirouzi et al. 2002), HCN (Chen et al. 2001; Decher et al. 2004), and spHCN1 (David et al. 2006) channels suggested that the S4-S5 linker couples S4 movement to channel opening and may stabilize the activation gate in a closed conformation outside the voltage activation range through an electrostatic interaction with the C-linker. The amino acids implicated in these interactions (R of the S4–S5 linker and D of the C-linker) are conserved in PIIH channels (Fig. 1B, in italic text). Insertion of segment BS, with its hydrophilic sequence of 3 amino acids, could change the interaction with C-linker, thereby favoring the open state of the channel; a similar effect is seen in the honey bee AMIHL variant (Gisselmann et al. 2004).
PIIH ABS-I, which contains both the S3–S4 segment A and the S4–S5 segment BS, produced more rapid activation kinetics and a more positive shift in activation than either insertion alone, indicating that their effects are largely additive. This suggests that the external S3–S4 linker and the cytoplasmic S4–S5 linker affect activation via independent mechanisms.
Variants which contain the longer segment BL in the S4–S5 linker domain did not express currents in oocytes. Segment BL contains an RKR ER retention motif that could keep the channels from trafficking to the surface. It also has a PKC phosphorylation site near the ER retention motif (Fig. 1B). Scott et al. (2001) reported that PKC phosphorylation of the C1 domain of the NR1 N-methyl-D-aspartate (NMDA) receptor suppresses a nearby RXR-type ER retention/retrieval motif, allowing expression of the receptor. PIIH splice variants containing BL may be regulated in their surface expression in the same way.
Splicing in the C-linker and CNBD regions and PIIH channel activation
The unbound cyclic nucleotide binding domain (CNBD) inhibits activation of Ih channels (Wainger et al. 2001). cAMP binding induces a conformational change that relieves the CNBD inhibition and facilitates channel opening (Wainger et al. 2001; Zagotta et al. 2003). Based on the X-ray crystal structures of the C-terminal fragments of HCN2 (Zagotta et al. 2003), our alternate variants C1 and C2 in the C-linker and CNBD region of PIIH are predicted not to affect the cAMP binding site. Alternate splicing here probably does not influence the affinity for cAMP but may change the efficacy of cAMP to affect channel opening. Consistent with this, replacement of C1 (in PIIH ABS-I) with C2 (in PIIH ABSC2-I) did not strongly affect channel activation parameters, but it did enhance cAMP's depolarizing shift in V1/2 (Fig. 5C).
Possible function of alternative splicing of PIIH channels with distinct properties in pyloric neurons
Peck et al. (2006) and Harris-Warrick et al. (1995) found that the biophysical parameters of Ih among the six cell types in the pyloric network vary in amplitude, Vact, and slope parameters. This variability is similar to that seen in our PIIH splice variants; for example, the activation V1/2 of PIIH splice variants varies between –107 and –82 mV in oocytes compared with endogenous variation between –103 and –85 mV in the different pyloric neurons. Could differential cell-specific expression of the splice variants underlie Ih heterogeneity in pyloric neurons? Single neuron RT-PCR will be necessary to address this question unambiguously. The H-currents evoked in Xenopus oocytes activated more quickly, and their slopes of activation were steeper than in the pyloric neurons. There are several possible explanations for these differences. The pyloric neurons' channels may be heterotetramers of various PIIH isoforms, as found in other HCN channels (Santoro et al. 2000; Ulens and Tytgat 2001), or could arise from a mix of independent homotetramers, each formed from a single splice variant. Alternatively, the neuronal channels may interact with auxiliary subunits that alter their properties (Qu et al. 2004; Yu et al. 2001) or undergo posttranslational modifications that are missing in oocytes. Finally, the neurons may have different baseline cAMP levels. Our results do not discriminate between these possibilities because the currents we measure in PIIH-injected PD neurons represent a mixture of endogenous and exogenous channels, and the proteins generated from the injected RNA might heteromerize with the endogenous channels.
We previously showed that in pyloric neurons the rate of postinhibitory rebound and the rebound spike frequency are co-regulated by Ih and IA (Harris-Warrick et al. 1995). Overexpression of IA, by injecting shal RNA into the pyloric neurons, failed to change the neurons' firing properties due to a homeostatic up-regulation of Ih (MacLean et al. 2003, 2005). In contrast, overexpression of a nonnative Ih gene, PAIH, did not alter the expression of IA, and this did lead to significant changes in firing properties of the PD neurons (Zhang et al. 2003). In the present study, we overexpressed two splice variants of the native P. interruptus gene, PIIH ABS-I and PIIH A-II, in PD neurons; these variants together express almost all of the significant sequence differences of functional PIIH splice variants from PAIH. Despite two- to ninefold increases in Ih, the amplitude and properties of IA were not significantly changed. These results support our earlier tentative conclusion that the homeostatic compensation between IA and Ih is unidirectional. However, it remains possible that overexpression of other PIIH splice variants containing segment D2, or variants with different combinations of alternate segments that we did not detect, might confer the ability to simultaneously upregulate IA.
As with PAIH (Zhang et al. 2003), overexpression of Ih did change the firing properties of the injected PD neurons but only when the maximal conductance was increased by more than two- to threefold (Fig. 7). It appears that Ih can only affect the pyloric motor pattern when a large number of channels can be activated in the normal voltage range. Even twofold overexpression of Ih in PD neurons did not significantly change the firing pattern of PD cells. Above this threshold increase in Ih, PD neuron firing parameters increased as a function of increasing Ih expression. There are several possible interpretations of this result. First, under normal conditions the endogenous Ih may play a rather limited role in shaping the firing properties of pyloric neurons as suggested by our recent work (Peck et al. 2006). Second, it is possible that most of the exogenously expressed Ih channels are inserted into the membrane in the soma, which is far away from the firing pattern generation sites located in the distal neuropil. We previously found this to be the case after injection of the transient potassium current gene, shal, into PD neurons (MacLean et al. 2005), so this is a real possibility. In both dynamic-clamp studies and a simple two-cell model, selectively raising Ih fourfold caused the neuron to depolarize, to phase advance and to fire additional spikes per burst (MacLean et al. 2005; Zhang et al. 2003). These modeling results mimic the effects of upregulating Ih through injection of PIIH.
In conclusion, the Ih gene of P. interruptus, PIIH, is extensively alternatively spliced to generate Ih channels with distinct activation kinetics, voltage dependence, and sensitivity to cAMP. Overexpression of two PIIH splice variants in PD neurons produced a large increase in Ih without altering the expression of IA, supporting our earlier conclusion that the homeostatic interaction between IA and Ih is unidirectional (Zhang et al. 2003). PIIH ABS-I overexpression altered the firing properties of the PD neurons but only when expression was above a minimal threshold and then in an Ih amplitude-dependent manner. Our study showed how well neurons can compensate for fluctuations in channel expression.
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Address for reprint requests and other correspondence: R. M. Harris-Warrick, Dept. of Neurobiology and Behavior, Cornell University, W159 Seeley G. Mudd Hall, Ithaca, NY 14853 (E-mail: rmh4{at}cornell.edu)
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) and after (
) application of 10 mM 8-Br-cAMP. C: shift of V1/2 by cAMP, calculated from B. D: time constants of activation (
) and PIIH ABS-I-expressing (
) PD neurons (n = 16 pairs of PDs). C: A-currents recorded in control and PIIH ABS-I-injected PD in the same ganglion. The cells were held at –50 mV, and after a 400-ms deinactivating prepulse to –120 mV, a series of 400-ms voltage steps were given from –50 to +40 mV in 10-mV increments. Insets: voltage protocols. D: peak conductance/voltage relationships for activation of IA in control (