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This Article Abstract Full Text (PDF) All Versions of this Article: 91/1/141    most recent expphysiol.2005.031773v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, M. Search for Related Content PubMed PubMed Citation Articles by Suzuki, M. Related Collections Symposia Papers Genomic Physiology

¹ Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical School 3311-1, Yakushiji, Minamikawachi, Tochigi, 329-0498, Japan

Abstract

Calcium-activated chloride currents (I_Cl(Ca)) can be recorded in almost all cells, but the molecular identity of the channels underlying this Cl^– conductance is still incompletely understood. Here, I report that tweety, a gene located in Drosophila flightless, possesses five or six transmembrane segments, and that a human homologue of tweety (hTTYH3) is a novel large-conductance Ca²⁺-activated Cl^– channel, while the related gene, hTTYH1, is a swelling-activated Cl^– current. hTTYH3 is expressed in excitable tissues, including the heart, brain and skeletal muscle, whereas hTTYH1 is expressed mainly in the brain. Expression of hTTYH3 in CHO cells generated a unique Cl^– current activated by an increase in the intracellular Ca²⁺ concentration. The hTTYH3-induced Cl^– current had a linear current–voltage (I–V) relationship, a large single-channel conductance (260 pS) and the anion permeability sequence I^– > Br^– > Cl^–. Like native Ca²⁺-activated Cl^– channels, the hTTYH3 channel showed complex gating kinetics and voltage-dependent inactivation, and was dependent on micromolar intracellular Ca²⁺ concentration. Expression in CHO cells of an hTTYH1 splice variant that lacks the C-terminal glutamate-rich domain of hTTYH1 (hTTYH1sv) generated a swelling-activated Cl^– current. I conclude that investigation of the tweety family will provide important information about large-conductance Cl^– channel molecules.

(Received 23 August 2005; accepted after revision 11 October 2005; first published online 11 October 2005)
Corresponding author M. Suzuki: Department of Pharmacology, Division of Molecular Pharmacology, Jichi Medical School 3311-1, Yakushiji, Minamikawachi, Tochigi, 329-0498, Japan. Email: macsuz{at}jichi.ac.jp

Chloride (Cl^–) is the most abundant anion and it passes across the cell membranes mainly through Cl^– channels. Cl^– channels are ubiquitously expressed and they play various roles, including stabilization of the membrane potential, transepithelial ion transport and cell volume regulation. Cl^– channels are thus variably regulated by voltage, calcium, pH and cell volume. Ca²⁺-activated Cl^– currents (I_Cl(Ca)) are widely observed in different cells and tissues. Three types of Ca²⁺-activated Cl^– channels have been reported: small (< 10 pS), intermediate (10–100 pS) and large (> 100 pS, maxi-Cl^–) conductance channels. Small and intermediate conductance Cl^– channels are found ubiquitously, even in oocytes, whereas large conductance Cl^– channels are rarer. Large conductance Cl^– channels are believed to transduce Ca²⁺ signals and stabilize membrane voltage (Jentsch et al. 2002).

Some information about the molecular identity of Ca²⁺-activated Cl^– channels has emerged recently. First, ClCA_1-4 (Gandhi et al. 1998; Gruber et al. 1998) were proposed to form intermediate conductance Cl^– channels when expressed in HEK cells. This I_Cl(Ca) is activated by a high concentration of Ca²⁺, is outwardly rectifying, and is inhibited by 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), dithiothreitol (DTT) and niflumic acid. However, hydrophobicity analysis shows that ClCA possesses one or two transmembrane segments (TMS), a structure that is unusual for the -subunits of ion channels. Therefore, the possibility that ClCA proteins activate endogenous Cl^– channels rather than being channels themselves cannot be excluded (Romio et al. 1999).

A second candidate Ca²⁺-activated Cl^– channel is bestrophin, the protein product of the gene defective in Best's disease (Marquardt et al. 1998). When expressed in HEK cells bestrophin encodes a small to intermediate conductance Ca²⁺-activated Cl^– channel (Sun et al. 2000). Human, Drosophila and nematode bestrophins (1 and 2) each possess four TMSs and generate different types of current–voltage (I–V) relationships. Furthermore, bestrophin mutants exert a dominant negative effect when coexpressed with wild-type bestrophin, suggesting that the functional bestrophin Cl^– channel is a homopolymer of wild-type subunits (Sun et al. 2000). While bestrophin encodes a small conductance Ca²⁺-activated Cl^– channel (Fischmeister & Hartzell, 2005) and ClCA_1-4 might regulate intermediate conductance Cl^– channels (Romio, 1999), the molecular identity of large-conductance Cl^– channels have until recently been obscure.

Cloning and structure of tweety gene products

To identify a novel ion channel family, I used a basic local alignment search tool (BLAST) to search for proteins with four or more TMSs predicted by hydrophobicity and a gene related to behaviour abnormality. After screening many proteins with a cluster of leucine residues as a transmembrane probe, I found a human gene (hTTYH3) that resembled Drosophila tweety (TTYH1) located in the flightless locus (Maleszka et al. 1996) and associated with a behavioural abnormality. The flightless gene is molecularly characterized by four transcription units, which are named tweety, fli, dodo and penguin. These genes are required for Drosophila to fly normally. The Drosophila mutant tweety is so called because, like the cartoon character, tweety cannot fly.

The gene htty3 is located at chromosome 7p22.3 and the protein hTTYH3 is identical to KIAA1691. Using BLAST, a family of genes related to htty3 were identified. These include CG3638 in Drosophila and two others in Homo sapiens (tty1 and tty2). Of note, disruption of CG3638 is lethal for Drosophila development. hTTYH3 mRNA encodes 4800 nucleotides and 523 amino acids. Although hTTYH1 is believed to have five TMSs (Campbell et al. 2000), hTTYH3 is predicted by the Sosui program (GenomNet, Kyoto University, Japan) to possess six TMSs (Fig. 1). The cluster of leucine residues used to identify hTTYH3 is found in TMS4.

View larger version (27K): [in this window] [in a new window]   Figure 1.  Proposed structure of the TTYH family Homology tree of TTYH family, including human and Drosophila TTYHs. Hydrophobicity was calculated by using Kyte-Doolittle with a window of 12. The lower schema shows the proposed structure. TTYHs encoding a large-conductance Ca2+-activated Cl– channel (left-hand two have 5 TMS or 6 TMS structures) in comparison to the structure of a large-conductance Ca2+-activated K+ channel, BKCa, found in Drosophilaslowpoke (right). Transmembrane segments (TMS1–6, cylinder), gylcosylation sites (red), pore region (P) with positively charged amino acids (blue circles) and E/D-rich C-terminal region (yellow) are illustrated.

The putative structure of hTTYH3 is comparable to that of the Ca²⁺-activated large-conductance K⁺ channel (Piskorowski, 2002). The pore region in TTYH3 can be predicted to be in a similar position to those of the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca), where positively charged amino acids, arginine (R) and histidine (H) are preserved in the TTYH family. BK_Ca has a unique aspartate (D) cluster below the last transmembrane domain. This negatively charged cluster is called a ‘Ca²⁺ bowl’ and is related to direct activation by Ca²⁺ (Schreiber et al. 1999). A Ca²⁺ bowl-like alignment is recognized in the TTYH family as a glutamate (E)- or aspartate (D)-rich domain in mammalian homologues that is capable of interacting with positively charged Ca²⁺ ions (Fig. 1). When compared with the structure of BK_Ca, TTYH3 lacks TMS3–4 and a voltage sensor. Therefore, the structure of TTYH3 predicts that it might encode a Ca²⁺-activated, voltage-independent large conductance Cl^– channel.

Distribution of TTYH3

TTYH3 is mainly distributed in excitable tissues. By Northern blotting, hTTYH3 mRNA (4.8 kb) was found in the brain, heart and skeletal muscle (Suzuki, 2004). Moreover, RT-PCR detected TTYH3 in mouse kidney, fat, thymus and uterus. When different cell lines were screened for the expression of TTYH3, TTYH3 was detected in an endothelial cell line (no. 375 cell from human aorta, Applied Cell Biology Research Institute, Washington, DC, USA), kidney epithelial cells (OK, HEK and MDCK) and a neuronal cell line (N2A). However, TTYH3 was not detected in Chinese hamster ovary (CHO) or human smooth muscle cells. Using an antibody that recognizes the C-terminus of TTYH3, positive staining was produced in thalamic neurones, ventricular cells of the heart, skeletal muscle membrane and mesangial cells in the glomerulus of the kidney.

Electrophysiological analysis of TTYH3

To investigate whether hTTYH3 encodes a large conductance Cl^– channel, the whole-cell patch-clamp technique was employed. Because large conductance channels have been detected in HEK293 cells (Zhu et al. 1998), hTTYH3 was expressed in CHO cells. Significant ionic conductance was not detected under control conditions. The Ca²⁺ ionophore ionomycin (1 µM) induced an outwardly rectifying current in mock transfected cells. However, in hTTYH3-transfected cells, ionomycin (1 µM) induced large linear currents (Fig. 2). The ionomycin-induced current in hTTYH3-transfected cells was unaffected by substitution of Na⁺ with tetraethylammonium (TEA⁺), but altered by substitution of Cl^– with gluconate. The permeability ratio (Hille, 1991) of hTTYH3 currents for Cs⁺/Cl^– was nominally zero, while that for gluconate/Cl^– was 0.33. These data suggest that the ionomycin-induced current in hTTYH3-transfected cells is Cl^– selective. Using a larger series of anions, the anion permeability (P_X/Cl) sequence of hTTYH3 was determined to be: I^– (2.2) > Br^– (1.8) > Cl^– (1) = SCN^– (1) > NO₃^– (0.83) > gluconate(0.3) = aspartate^– (0.2) = Na⁺ (< 0.1).

View larger version (25K): [in this window] [in a new window]   Figure 2.  Whole-cell currents of TTYH3 in CHO cells A, representative currents were recorded from –100 to +90 mV in steps of 10 mV from unstimulated (left) and ionomycin-treated (1 µM; centre) control CHO cells and hTTYH3-transfected CHO cells treated with ionomycin (1 µM; right). B, top, amino acid sequences of the putative pore region of hTTYH1–3. Positively charged amino acids R366 and H370 in hTTYH3 are conserved and mutated. Bottom, I–V relationships of wild-type and mutant hTTYH3s in the presence of the indicated ions.

To explore the pharmacological profile of hTTYH3, the effects of Cl^– channel blockers on hTTYH3 whole-cell Cl^– currents were investigated. Niflumic acid (300 µg ml^–1) blocked the ionomycin-evoked Cl^– current in non-transfected CHO cells, but was without effect on hTTYH3 Cl^– currents. Similarly, hTTYH3 Cl^– currents were not inhibited by DTT (10 µM), ZnCl₂ (10 µM) or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS; 10 µM). However, hTTYH3 Cl^– currents were completely inhibited by DIDS (10 µM).

Single-channel studies demonstrated that hTTYH3 had properties consistent with those of the maxi-Cl^– channel, including a linear I–V relationship in symmetrical Cl^– rich solutions with a slope conductance of 260 pS (Fig. 3). Analysis of current amplitude histograms revealed a subconductance of 50 pS. hTTYH3 Cl^– channels exhibited complex gating behaviour, with long channel openings observed at +60 mV, but brief, flickery openings at –60 mV (Fig. 3). The open probability of hTTYH3 was voltage independent at negative voltages, but showed some voltage dependence at large positive voltages. Like the BK_Ca channel (Schreiber, 1999), a high concentration of cytosolic Ca²⁺ was essential for hTTYH3 single-channel activity (effective dose (ED)₅₀, 2 µM). Taken together, the electrophysiological characteristics of hTTYH3 demonstrate that it is a bona fide maxi-Cl^– channel.

View larger version (13K): [in this window] [in a new window]   Figure 3.  Single hTTYH3 channel currents A, representative single-channel traces at the indicated voltages. Continuous and dotted lines show the open and closed states, respectively. B, single-channel I–V relationship of hTTYH3. Data are means ± S.E.M. (n = 12). C, current amplitude histogram of hTTYH3 at +40 mV. Gaussian analysis shows two conductances: 50 and 260 pS.

hTTYH1 cDNAs containing C-terminal spliced variants were identified. One variant (hTTYH1sv) isolated from a retinal library has the C-terminal E-rich domain replaced with a different C-terminal. Northern blot analysis suggested that hTTYH1 is expressed mainly in the brain. To elucidate whether TTYH1 and TTYH1sv may coexist, RT-PCR was performed on isolated mouse tissues. mTTYH1, but not mTTYH1sv, was detected in hippocampus and hypothalamus, while mTTYH1sv, but not mTTYH1, was found in dorsal root ganglia. These data suggest that TTYH1sv might function independently of the presence of TTYH1.

In contrast to hTTYH3, hTTYH1sv was not activated by an increase in the intracellular Ca²⁺ concentration. Instead, hTTYH1sv was activated by cell swelling (Fig. 4). Under isotonic conditions no whole-cell current was detected in hTTYH1sv-expressing CHO cells. However, reducing the osmolarity of the extracellular solution to either 250 or 220 mosmol l^–1 activated large linear currents that were inhibited by GdCl₃ (50 µM) and DIDS (10 µM; Fig. 4). hTTYH3-expressing CHO cells generated GdCl₃-sensitive whole-cell currents in the presence of a 220 mosoml l^–1 extracellular solution (Fig. 4). However, the magnitude of activated hTTYH3 Cl^– current did not differ from that of GFP-transfected control CHO cells (Li et al. 2000).

View larger version (22K): [in this window] [in a new window]   Figure 4.  Effects of hypotonic solution on hTTY1sv and hTTY3 currents A, whole-cell currents were recorded with CsCl and without CaCl2 in the intracellular solution from –100 to +90 mV in steps of 10 mV in hTTY1sv-transfected (upper panel) and hTTY3-transfected CHO cells (lower panel). The extracellular solution was changed to hypotonic solutions of 250 and 220 mosmol l–1. B, the magnitude of the current at 100 mV was measured in six experiments with hTTY1sv current (left panel) and with hTTY3 current (right panel). Significant increase (ANOVA) compared with the control (290 mosmol l–1) is indicated by *P < 0.05, **P < 0.001. 220 + GdCl3 and 220 + DIDS indicate that 50 µM of GdCl3 and 10 µM of DIDS were dissolved in 220 mosmol l–1 solution, respectively. hTTY3(–) indicates the endogenous current of CHO cells exposed to solutions of 290 and 220 mosmol l–1.

The I_Cl(Ca) induced by hTTYH3 shows characteristics similar to those of native maxi-Cl^– channels. First, most Cl^– channels are blocked by niflumic acid or NPPB at a concentration of 300 µg ml^–1. The maxi-Cl^– channel is, however, not blocked by niflumic acid, but by DIDS (Kokubun et al. 1991; Taylor & Roper, 1994). Second, voltage-dependent suppression at large depolarized voltages has been observed in some Ca²⁺-activated Cl^– channels (Thorn & Martin, 1987; Coulombe & Coraboeuf, 1992). Third, Ca²⁺-activated Cl^– channels in muscle cells have complex conduction mechanisms (Coulombe, 1992) and gating behaviour (Hussy, 1992). Fourth, the hTTYH3 channel displayed a permeability sequence of I^– > Br^– > Cl^–, which is observed in skeletal muscle (Dixon et al. 1993) and corresponds to Eisenman's sequence 1. The ionic environment within the channel is, however, influenced by the cationic environment, which alters the selectivity of Cl^– channels (Wright & Diamond, 1977), suggesting that this order of permeability might not be specific for the hTTYH3 structure. Although volume-sensitive large-conductance Cl^– channels are rarely described (Bell et al. 2003; Sabirov et al. 2001), they may be important for regulating body fluid homeostasis.

An ion channel with six TMSs is a prototype structure of cation-permeable channels, such as the K⁺ channel encoded by the K_v family and the Ca²⁺ channel encoded by the Trp family (Minke & Cook, 2002). K_v and Trp channels were identified based on the abnormal behaviour of Drosophila mutants. Thus, based on the data summarized in this review, the TTYH family might also become a prototype Cl^– channel.

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Acknowledgements

We would like to thank Yuki Oyama and Yuko Watanabe for technical assistance and Jyunichi Taniguchi for critical discussion. Professor Suzuki's participation in the Physiology of Anion Transport meeting was supported by a grant from the The Great Britain Sasakawa Foundation.

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/1/141    most recent expphysiol.2005.031773v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suzuki, M. Search for Related Content PubMed PubMed Citation Articles by Suzuki, M. Related Collections Symposia Papers Genomic Physiology