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First published online January 31, 2006
Journal of Experimental Biology 209, 731-747 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02032

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A potassium channel (Kv4) cloned from the heart of the tunicate Ciona intestinalis and its modulation by a KChIP subunit

Vicenta Salvador-Recatalà^1,2, Warren J. Gallin³, Jennifer Abbruzzese⁴, Peter C. Ruben⁴ and Andrew N. Spencer^2,3,*

¹ Department of Physiology, University of Alberta, Edmonton, AB, T6G 2H7, Canada
² Bamfield Marine Sciences Centre, Bamfield, BC, V0R 1B0, Canada
³ Department of Biological Sciences, University of Alberta, Edmonton, AB, T6G 2E9, Canada
⁴ Department of Biology, Utah State University, Utah, 84322 USA

View larger version (18K): [in a new window]   Fig. 1. RT-PCR amplification of the 3' ends of mRNAs that encode Kv4 and KChIP, extracted from heart tissue of C. intestinalis. (A) Fragments of 1500 bp corresponding to the 3' end of the transcript for a Kv4 channel, amplified by 3'-RACE. No cDNA was added to the control reaction. (B) Diagram showing the organization of the CionaKv4 gene, based on an alignment between the transcript sequence for CionaKv4 and scaffold 168 of the genomic database for C. intestinalis (release version 1.0). Exons are shown as black boxes and numbered in Arabic numerals (0-6). Introns are shown as white boxes and numbered in Roman numerals (I-VI). Approximate intron sizes (in bp) are indicated above the introns. Exon sizes (in bp) are indicated below the boxes. Exon sizes, but not intron sizes, are drawn approximately to scale. The 3'-UTR region is represented by a box shaded in grey. The box representing the 5'-UTR region is delimited by a discontinuous line to indicate that the sequence of this region was not determined in the present study. (C) Fragments of 600 bp, corresponding to the 3' end of the transcript for a KChIP subunit, amplified by 3'-RACE. No cDNA was added to the control reaction. (D) Diagram showing the exon/intron structure of the CionaKChIP gene, as derived from an alignment between the sequence of the CionaKChIP transcript and scaffold 457 of the genomic database for C. intestinalis (release version 1.0). Exons are shown as black boxes and numbered in Arabic numerals (1-7). Introns are shown as white boxes and numbered in Roman numerals (I-VI). Intron and exon sizes (in bp) are indicated, respectively, above or below the diagram and are drawn approximately to scale. The 3'-UTR region is represented by box shaded in grey. The 5'-UTR region is delimited by a discontinuous line to indicate that its sequence was not determined in this study.

View larger version (85K): [in a new window]   Fig. 2. Alignment and sequence comparison of Kv4 channels from diverse metazoans. Alignment of the deduced amino acid sequence of the three mammalian Kv4 isoforms (Kv4.1, Kv4.2 and Kv4.3), their tunicate homologues (CionaKv4 and TuKv4), lobster Shal (lShal) and jellyfish Shal (jShal). T-Coffee software (Notredame et al., 2000) was used to align these sequences, with the following GenPept. accession numbers: CionaKv4, AAS00646; TuKv4, BAC53863; Kv4.1, 27436981; Kv4.2, 9790093; Kv4.3, 6653655; lShal, AAA81592; jShal, AAB39750. Residues that are identical for at least four of the seven channels are shown in reverse lettering (white on black). Membrane-spanning domains S1-S6, the pore region and the N terminus are underlined. Arrows indicate exon/intron boundaries for CionaKv4 only. The di-leucine motif is labelled. Putative phosphorylation sites for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) are indicated by filled circles and open circles, respectively.

View larger version (44K): [in a new window]   Fig. 3. Alignment and sequence comparison between CionaKChIP and representatives of the four vertebrate KChIP isoforms. The deduced amino acid sequence of CionaKChIP and selected KChIPs belonging to the four mammalian isoforms were aligned using T-Coffee software (Notredame et al., 2000). KChIP subunits and GenPept. accession numbers were: CionaKChIP, AAS00647; KChIP1a, AAL12489; KChIP2a, AAF81755; KChIP3, Q9Y2W7; KChIP4b, NP_079497. Residues that are identical for at least three of the five KChIPs are shown in reverse lettering (white on black). The positions of the four EF-hands are underlined and labelled below the alignment. X,Y,Z,-Y,-X,-Z are the residues that coordinate Ca2+ (Bairoch and Cox, 1990). Arrows indicate exon/intron boundaries for CionaKChIP only. Putative phosphorylation sites for protein kinase C (PKC) are indicated by filled circles.

View larger version (32K): [in a new window]   Fig. 4. Phylogenetic relationships of CionaKv4 and CionaKChIP. (A) Phylogenetic relationships of Kv4 channels from different species. Two cnidarian Kv4 channels were used as an out-group to polarize the relationships of the other channels. The two arthropod channels, Fly Shal and Lobster Shal, group together as a sister group to the chordate channels. Shal and Ciona are indicated in bold type. The two tunicate channels, from Halocynthia roretzi and Ciona intestinalis, group together and are basal to the clade containing all three paralogues of the vertebrates. All of the vertebrate channels group within one of three paralogous clades, indicating that the three vertebrate Kv4 paralogues were present in the common ancestor of all vertebrates, but not in the common ancestor of vertebrates and tunicates. (B) Phylogenetic relationships of KChIPs from different species. Two arthropod KChIPs that were found in BLAST searches of the genomes of Drosophila (fly) and Anopheles gambiae (mosquito) were used as an out-group to polarize the relationships of the other KChIPs. Numbers above or below the lines indicate Bayesian posterior probability, calculated with the MrBayes program. Unlabelled nodes have a posterior probability of 1. GenPept. accession numbers of the protein sequences are indicated in parentheses. The scale bars represent a divergence equivalent to an average of a 10% change in amino acids.

View larger version (41K): [in a new window]   Fig. 5. Currents produced by CionaKv4 and an N-terminal deleted mutant of CionaKv4 (ntCionaKv4) in the presence and absence of CionaKChIP. (A) Representative currents from CionaKv4 channels expressed alone. (B) Currents produced by CionaKv4 co-expressed with CionaKChIP. (C) Currents produced by ntCionaKv4 channels expressed alone. (D) Currents produced by ntCionaKv4 co-expressed with CionaKChIP. All recordings were obtained using the macro-patch technique. Currents were evoked by depolarizing the macro-patches for 0.5 s from a holding potential of -100 mV to +80mV in 10 mV steps. The form of the stimulus protocol is given on the

View larger version (39K): [in a new window]   Fig. 6. Activation properties of CionaKv4 and an N-terminal deletion mutant of CionaKv4 (ntCionaKv4) in the presence and absence of CionaKChIP. (A) Comparison between current-voltage relationships for CionaKv4 alone and in the presence of CionaKChIP (Ai), CionaKv4 and ntCionaKv4 (Aii), and ntCionaKv4 alone or with CionaKChIP (Aiii). N=17. (B) Comparison between the time constants of macroscopic activation ( activation)-voltage relationships for CionaKv4 alone or in the presence of CionaKChIP (Bi), for CionaKv4 and ntCionaKv4 (Bii), and for ntCionaKv4 alone or with CionaKChIP (Biii). Solid curves represent single exponential fits to these relationships. N=12. (C) Comparison between normalized peak conductance-voltage relationships for CionaKv4 alone or with CionaKChIP (Ci), for CionaKv4 and ntCionaKv4 (Cii), and for ntCionaKv4 alone or with CionaKChIP (Ciii). N=17. Solid curves represent first order Boltzmann fits of the averaged data. ntCionaKv4 symbolizes CionaKv4 channels lacking N-terminal amino acids 2-32. Values are means ± s.e.m.

View larger version (34K): [in a new window]   Fig. 7. Inactivation properties of CionaKv4 and an N-terminal deletion mutant of CionaKv4 (ntCionaKv4) in the presence/absence of CionaKChIP. (A) Comparison of the time constant of macroscopic inactivation ( inactivation)-voltage relationships for (Ai) CionaKv4 alone (wt) or in the presence of CionaKChIP (wt+), (Aii) CionaKv4 (wt) and ntCionaKv4 (nt), and (Aiii) ntCionaKv4 alone (nt) or with CionaKChIP (nt+). 1 (circles) and 2 (squares) are the time constants of the fast and slow components of inactivation, respectively, of CionaKv4 (solid symbols) or ntCionaKv4 (open symbols) currents. (diamonds) is the time constant of the single component of inactivation of CionaKv4/KChIP (solid diamonds) or ntCionaKv4/KChIP (open diamonds). All inactivation kinetics appear to be insensitive to voltage. N=15. (B) Comparison of the time constant of deactivation ( deactivation)-voltage relationships for (Bi) CionaKv4 alone or in the presence of CionaKChIP, (Bii) CionaKv4 and ntCionaKv4, and (Biii) ntCionaKv4 alone or with CionaKChIP. N=16. (C) Comparison of the steady-state inactivation curves for (Ci) CionaKv4 alone or with CionaKChIP, (Cii) CionaKv4 and ntCionaKv4, and (Ciii) ntCionaKv4 alone or with CionaKChIP. Solid curves represent first order Boltzmann fits of the averaged data. Steady-state inactivation was determined by measuring the peak current evoked with a depolarizing pulse to +50 mV as a function of the voltage of a preceding 10 s prepulse test (between -130 and -30 mV). N=8. (D) Comparison of the rates of recovery from inactivation for (Di) CionaKv4 alone or in the presence of CionaKChIP, (Dii) CionaKv4 and ntCionaKv4, and (Diii) ntCionaKv4 alone or in the presence of CionaKChIP. N=8. A double-pulse protocol was used with a test pulse to +50 mV lasting 1 s separated by a recovery period (at -100 mV) of increasing duration (50-2000 ms) from a second test pulse to +50 mV. The currents evoked by the second pulse (I0) were normalized to the currents produced by the first pulse (I) and plotted against the duration of the interpulse interval. Solid curves represent single exponential fits to the data. Values are means ± s.e.m.

View larger version (22K): [in a new window]   Fig. 8. Alignment and sequence comparisons between the N termini of CionaKChIP and representatives of each mammalian KChIP isoform. (A) Alignment between the N termini of CionaKChIP and KChIP4a (GenPept. accession number AAL86766), which contains the K inactivation suppressor domain (KIS). (B) Alignment between the N termini of CionaKChIP and KChIP1a (GenPept. accession number AAL12489). (C) Alignment between the N termini of CionaKChIP and KChIP2a (GenPept. accession number AAF81755). (D) Alignment between the N termini of CionaKChIP and KChIP3a (GenPept. accession number Q9Y2W7). T-Coffee software (Notredame et al., 2000) was used to align these sequences. Residues that are identical for each pair of sequences are boxed.