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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY
Faculty of Biosciences, University of Joensuu, Joensuu, Finland
Submitted 23 August 2007 ; accepted in final form 29 April 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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fish heart; potassium current; temperature acclimation; cardiac action potential; excitation-contraction coupling
-subunits encoded by the ether-à-go-go-related gene (ERG or KCNH2) (38). Each ERG -subunit consists of six -helical transmembrane domains and a pore-forming P-loop with the signature sequence (GFGN) of the K+ selectivity filter. An accessory β-subunit mink-related peptide 1 coassembles with ERG and is supposed to be crucial for the formation of the native IKr (1), although there is no direct evidence for an association between mink-related peptide 1 and ERG in cardiac myocytes. Distinct time- and voltage-dependent kinetics of the ERG channel, characterized by delayed activation and rapid inactivation during depolarization and fast recovery from inactivation and slow deactivation on repolarization (26, 24, 28), determine IKr amplitude during different phases of the cardiac AP (33). Physiologically, this appears as a small repolarizing current (strong inward rectification) at positive membrane potentials of the early plateau phase and results in larger outward current flow when AP begins to repolarize.
ERG channels are synthesized in endoplasmic reticulum (ER). In the ER, immature ERG proteins are modified by glycosylation (42) and guided to the correct three-dimensional folding pattern by cytosolic chaperones (13). Both events are important for the trafficking of ERG proteins within the cell and can significantly affect channel density in the plasma membrane. At low temperatures, ERG proteins stay longer in the ER, which increases the number of correctly folded proteins and thus ERG channel density in the sarcolemma (11, 30). Furthermore, ERG channel function is regulated in a complex manner by protein kinase-mediated phosphorylation and interactions with membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) (8). Clearly, IKr density is an outcome of many interacting factors, including ERG channel structure, its association with regulatory β-subunit, temperature-dependent trafficking to plasma membrane, lipid membrane composition, and covalent modification by intracellular regulators. Each of those factors could contribute to the IKr phenotype in vertebrate cardiac muscle under various physiological conditions.
Body temperature of ectothermic animals can vary considerably, e.g., due to seasonal changes in ambient temperature. To maintain adequate physical activity and rate of body functions relatively independent from temperature changes, many fish species are able to recruit compensatory mechanisms during prolonged exposure to altered temperature (14, 29). In rainbow trout (Oncorhynchus mykiss, Walbaum), excitability of cardiac myocytes is improved during chronic cold exposure through compensatory increases in the density of sodium and potassium currents. In particular, AP duration is shortened via upregulation of the cardiac IKr, and the same current seems to be involved in increasing heart rate in the cold-acclimated trout (37, 16). The objective of this study was to examine underlying molecular mechanisms of temperature-induced differences in the IKr density of the rainbow trout heart. Specifically, we hypothesized that differences in IKr current density are due to increased expression of ERG channels or temperature-related changes in gating kinetics, leading to increased K+ conductance in the cold. To this end, we cloned the rainbow trout ERG (omERG) gene, determined its regional and temperature-dependent expression in the heart, and examined electrophysiological properties of the IKr current in atrial myocytes of thermally acclimated rainbow trout.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Rainbow trout (body mass 290.0 ± 67.9 g, n = 13) were obtained from a local fish farm (Kontiolahti, Finland). In the laboratory, the fish were reared in temperature-controlled 500- or 1,000-liter stainless tanks with a continuous supply of aerated groundwater (
0.5 l/min) and were acclimated to 4°C [cold acclimation (CA)] or 18°C [warm acclimation (WA)] under a 15:9-h light-dark photoperiod for at least 4 wk before use in experiments. Fish were fed commercial trout food (Biomar, Brande, Denmark) to satiation five times a week. All experiments were done with the permission of the national committee for animal experimentation.
Molecular Methods
Extraction of RNA. Fish were stunned by a sharp blow to the head and killed by cutting the spine. Atrium and ventricle were quickly removed and frozen immediately in liquid nitrogen. Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. RNA was quantified by UV-spectrophotometer and qualified by agarose gel electrophoresis.
Cloning of omERG with RT-PCR. First-strand cDNA was prepared from DNase-treated cardiac total RNA using random hexamers (Promega, Madison, WI) and M-MuLV Reverse Transcriptase RNase H- (Finnzymes, Espoo, Finland). Degenerative primers (Table 1) were used to clone partial cDNA fragments of the ERG gene under PCR conditions, described by Hassinen et al. (15). New forward and reverse primers were designed on the basis of the obtained sequences for further cloning. The 5'- and 3'-RACE kits (Invitrogen) were used to clone the 5'- and 3'-untranslated region (UTR) of the gene, respectively. All PCR products were analyzed by gel electrophoresis, extracted from gel by Qiaex II Gel Extraction Kit (Qiagen, Valencia, CA), and cloned to the pGEM-T Easy vector (Promega). At least one clone was sequenced using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The reliability of the sequences was also rechecked with direct sequencing from the purified PCR product.
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In situ hybridization.
To prepare RNA probes for the omERG gene, a DNA fragment containing nucleotides 268–1,023 was ligated to the pGEM-T-Easy vector. Sense and antisense RNA probes were transcribed by T7 and SP6 RNA polymerases, respectively, and labeled with digoxigenin, according to the manufacturer's instructions (Roche Biochemicals, Mannheim, Germany). Probes were treated by alkaline hydrolysis to final length of 250 bp and quantified by a dot plot method, according to the manufacturer's instructions.
Excised hearts were quickly placed in fresh 4% paraformaldehyde in phosphate buffered saline. Tissues were fixed overnight at 4°C, dehydrated with increasing ethanol series, cleared in xylene, and embedded in paraffin. Seven-micrometer-thick tissue sections were cut and attached on Menzel Superfrost Plus slides (Menzel Gläser, Braunschweig, Germany), so that consecutive sections could be hybridized with sense and antisense probes. The sections were deparaffinized with xylene, rehydrated in decreasing ethanol series, and pretreated as described by Di Laurenzio et al. (12). Hybridization was carried for 16 h in 50% formamide atmosphere at 45°C, and thereafter the unbound probe was removed by RNase A treatment. Hybridization was visualized by anti-digoxigenin-alkaline-phosphatase conjugate and Dig Nucleic Acid Detection Kit, according to the manufacturer's instructions (Roche Biochemicals). Finally, slides were dehydrated in ethanol, cleared in xylene, mounted with Depex (Merck Eurolab, Darmstadt, Germany), and sealed with coverslips. Zeiss Axioplan II Imaging microscope and Zeiss Axiocam HR digital camera were used in photographing.
Electrophysiological Methods
Myocyte isolation. Atrial myocytes were isolated by enzymatic digestion using trypsin and collagenase (15, 35) and were used within 8 h from isolation.
Patch-clamp experiments.
Whole cell voltage-clamp experiments were performed using an Axopatch 1-D (Axon Instruments) or an EPC-9 (HEKA Instruments, Lambrecht/Pfalz, Germany) amplifier. A small aliquot of myocyte suspension was transferred to a recording chamber and superfused with external saline solutions at the rate of 1.5–2.0 ml/min. The experiments with myocytes of CA animals were conducted at 4 and 11°C, and those with WA animals at 11 and 18°C. Consequently, results were obtained both at the physiological body temperatures of the animals and at the common experimental temperature of 11°C, the latter enabling direct comparison of results between the acclimation groups. Temperature was adjusted to the desired value by using two circulating water baths or a Peltier device (TC-10, Dagan, Minneapolis, MN) and was continuously monitored by a thermistor positioned close to the myocyte. Patch pipettes were pulled from borosilicate (Garner, Claremont, CA) using a vertical two-stage puller (List-Medical, L/M-3P-A, Darmstadt, Germany). Off-set potentials were zeroed before the formation of gigaohm seal, and the pipette capacitance (8.96 ± 0.20 pF, n = 50) was compensated after the seal formation. Cell membrane was pierced by a short-voltage pulse, and capacitive transient was eliminated by adjusting series resistance and cell membrane capacitance compensation circuits. Electrode resistance and total access resistance were 2.50 ± 0.08 and 7.70 ± 0.49 M
(mean ± SE, n = 50), respectively.
Solutions. A nominally Ca2+-free saline used in myocyte isolation contained the following (in mmol/l): 100 NaCl, 10 KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose, and 10 HEPES, pH 6.9 at 20°C. For enzymatic digestion, 0.75 mg/ml collagenase (Type IA, Sigma), 0.5 mg/ml trypsin (Type IX, Sigma), and 0.5 mg/ml fatty-acid-free bovine serum albumin (A6003, Sigma) were added to this saline. The external saline solution contained (in mmol/l): 150 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.7 with NaOH). In addition, tetrodotoxin (0.5 µM; Tocris Cookson), nifedipine (10 µM; Sigma), and glibenclamide (10 µM; Sigma) were added to the solution to block Na+, Ca2+, and ATP-sensitive K+ current, respectively. The pipette solution contained the following (in mmol/l): 140 KCl, 1 MgCl2, 5 EGTA, 4 MgATP, and 10 HEPES (pH adjusted to 7.2 with KOH).
Steady-state activation and inactivation. Voltage dependence of steady-state activation was obtained by a voltage protocol, where membrane potential was clamped from the holding potential of –40 mV to test potentials between –80 and +80 mV in 20-mV increments to activate the channels. Potassium efflux through the activated omERG channels at constant driving force was obtained from peak tail currents elicited on return to –40 mV. Normalized tail currents (I/Imax, where I is current and Imax is maximum current) were plotted as a function of the prepulse voltage and fit to the Boltzmann equation:
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where V is membrane potential, V0.5 is the midpoint, and S is the slope of the curve.
Steady-state inactivation (inward rectification) was measured by a double-pulse protocol. Channels were first activated and inactivated by depolarization to +20 mV and then allowed to recover from inactivation by clamping membrane potential between –120 and +40 mV. Peak tail currents at the beginning of the test pulse are proportional to the number of channels recovered from inactivation. Since channels start to deactivate at the beginning of the tail pulse, error due to deactivation was corrected by extrapolation of tail currents to the beginning of the pulse. The relative proportion of inactivated IKr was obtained by dividing the measured tail current with the noninactivated current, which was predicted from the linear portion of the current between –120 and –80 mV. To obtain steady-state voltage dependence of inactivation, this current was blotted as a function of the tail pulse potential and fit to the Boltzmann distribution with a negative slope (–S).
Whole cell conductance of IKr. A triple-pulse voltage protocol was used to get channels in open conducting state. To fully activate and inactivate ERG channels, membrane potential was first clamped from the holding potential of –40 to +20 mV. Next, the membrane potential was hyperpolarized to –120 mV for 5–20 ms (depending on temperature) to allow recovery of channels from inactivation and thus induction of an open-channel configuration. The current flow through fully open channels was obtained by measuring the tail current amplitude at the beginning of test pulses ranging from –120 to +40 mV. To exclude the effect of inactivation on IKr, tail currents were fitted with monoexponential equation, and the current value was read from the fit at the start of the test pulse. (The rate of onset of inactivation between 0 and +60 mV was also obtained from these fits; see below.) The whole cell conductance (nS/pF) of the IKr was obtained by dividing the linear current (pA) between –120 and –40 mV with the voltage gradient (160 mV) and by normalizing to the cell size (pF).
Kinetics of activation, deactivation, and inactivation. Activation time course was estimated using the envelope of tails method. Membrane potential was depolarized from –40 to 0 mV for 100–3,600 ms, and tail currents elicited on return to –40 mV were plotted as a function of the prepulse duration and fit with a sigmoid equation to obtain half-time for activation. Deactivation kinetics were measured as function of membrane voltage between –120 and –40 mV by tail currents elicited after 3,000-ms depolarization from –40 to 0 mV. Deactivating tail currents were fit with a single exponential function (y = a–bt; where a is current, t is time, and b is the time constant of the function) and plotted against test pulse voltages.
Temperature dependence of IKr kinetics was expressed as a Q10 value and was obtained from the equation
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where T1 and T2 are the temperatures that produce the kinetic rates of R1 and R2, respectively.
Statistics
Effects of thermal acclimation and acute temperature changes on IKr were tested using Student's t-test for paired or unpaired samples. Nonparametric Mann-Whitney or Kruskal-Wallis test was used after logarithm conversion of the data, if data were not normally distributed. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Inward rectification and repolarizing power of the IKr are dependent on time- and voltage-dependent kinetics of ERG channels. To this end, we examined the effect of temperature acclimation on kinetic properties of the trout atrial IKr. Time dependence of the IKr activation was measured as the peak tail current following a voltage step from –40 to 0 mV for 100 to 3,600 ms (Fig. 1). At 11°C, the time for half-maximal IKr activation was similar in CA (647 ± 70.5 ms) and WA trout (851 ± 78.4 ms) and about twice as long in CA trout at 4°C (720.5 ± 48.9 ms) as in WA trout at 18°C (378.0 ± 41.0 ms) (P < 0.05), suggesting that thermal acclimation does not affect activation kinetics of the omERG. It should be noted, however, that temperature dependence of activation was much less in CA trout between 4 and 11°C (Q10 = 1.16) than in WA trout between 11 and 18°C (Q10 = 3.18).
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To study voltage dependence of steady-state activation, omERG channels were activated by depolarizing steps from –40 mV to potentials between –80 and +80 mV, and the activation was measured from tail currents upon return to –40 mV (Fig. 4A). Normalized tail current amplitude was plotted against the voltage of the preceding depolarizing step. Voltage dependence of activation was fully resistant to temperature changes. Neither temperature acclimation nor acute temperature changes affected half-voltage (V0.5) of activation. Accordingly, V0.5 of steady-state activation was similar in CA (–18.1 ± 1.2 mV at 4°C) and WA trout (–16.4 ± 0.9 mV at 18°C) at the physiological body temperatures of the fish (Fig. 4B). In contrast, the S of the Hill curve was steeper in CA (7.1 ± 0.3) than WA trout (11.0 ± 0.8) (P < 0.05), when measured at 11°C. However, this difference disappeared at physiological body temperatures of the fish (9.6 ± 0.5 and 9.5 ± 1.3 for CA and WA fish, respectively), indicating thermal compensation of the Hill S upon CA.
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Whole Cell Conductance of the IKr
Although thermal resistance of steady-state activation and inactivation, temperature compensation of Hill S of activation, and low thermal sensitivity of activation kinetics of the CA trout may account to some extent for the differences in IKr between WA and CA fish at physiological temperatures, these variables were indistinguishable for CA and WA trout at 11°C and, therefore, cannot explain the larger IKr density of the CA trout at this temperature (37). To clarify this issue, we measured whole cell conductance of the IKr, which is the product of single-channel current amplitude (i) and the number (N) of active channels (I = i * N), and, therefore, informs about the number of active ERG channels. Whole cell conductance of the fully activated IKr was measured using a triple-pulse protocol, where IKr was first activated and inactivated at +20 mV, then released from inactivation during a short hyperpolarizing pulse to –120 mV, and finally conductance of the fully activated channels was measured at voltages ranging from –120 to +40 mV (Fig. 5). Current-voltage relation of the fully activated channels was linear and had a reversal potential at about –75 mV, close to the equilibrium potential of K+ ions. At 11°C, the whole cell conductance was 1.91 times as high in CA (0.21 ± 0.01 nS/pF) as in WA (0.11 ± 0.01 nS/pF) (P < 0.05) trout, and, at physiological body temperatures of the fish, the difference completely disappeared (Fig. 5). Q10 values for the whole cell conductance were 1.36 for CA fish (4–11°C) and 1.31 for WA fish (11–18°C). These findings indicate a complete thermal compensation of the whole cell conductance of the trout atrial IKr and strongly suggest that the cold-induced increase in current density is caused by an increase in the number of functional ERG channels.
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omERG (GenBank accession number EF689136) cDNA was cloned by RT-PCR and consisted of a 5'-UTR of 75 bp, an open-reading frame of 3,339–3,594 bp encoding 1,113–1,198 amino acids, and a 3'-UTR of 36–487 bp (Fig. 6; Supplemental Fig. 1). (The online version of this article contains supplemental data.) Deduced protein sequence of the omERG showed high similarity with other species' ERG proteins. Coding sequence of the omERG shared nucleotide identity of 70% with zebrafish and 65% with mammalian ERG, corresponding to 78 and 67% similarity at the protein level, respectively. Considerable variation, including 14 differentially processed 3' ends, was found in the 3' end of the omERG cDNA. In addition, 17 nucleotide substitutions, 9 of which led to amino acid differences, were found from the coding sequence of the omERG (Supplemental Fig. 1). Transmembrane domains (S1-S6), pore loop (P), Per-Arnt-Sim (PAS) domain, followed with PAS-associated COOH-terminal motif and cyclic-nucleotide-binding domain were identified by amino acid alignment with other species' ERG genes and showed much higher conservation than NH2- and COOH-terminal cytoplasmic tails. Two asparagine (N)-linked glycosylation consensus sites (N-X-T/S, where X is any residue except proline), located in the S5-S6 linker of the mammalian ERG, exist also in the omERG. Putative phosphorylation sites of the omERG protein were scanned using PROSITE software. Together, 21 consensus sequences for PKA- and PKC-mediated phosphorylation were found in the omERG, which markedly differ from the 18 phosphorylation sites of the mammalian ERG, only three of them being similar. Furthermore, ER retention signal (R-G-R) and two cell attachment sequences (R-G-D) of the mammalian ERG protein were lacking in the omERG.
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Since electrophysiological analyses suggest that a large part of the difference in IKr density between CA and WA trout is due to a higher number of omERG channels in the CA trout, transcript abundance of the omERG gene was studied from atrium and ventricle of the trout heart. Examination of regional distribution of the omERG transcripts by in situ hybridization shows that omERG is expressed throughout the muscular tissue of the trout heart (Fig. 7). It is absent from bulbus arteriosus and sinus venosus, but evenly expressed in atrial muscle and also present in sinoatrial nodal tissue and the sinoatrial valve, although in the latter two tissues in lesser amounts than in the atrium proper. In the ventricle, omERG transcript density seems to be higher in the outer compact epicardium than in the inner spongious endocardium (Fig. 7). However, control sections treated with the sense probe indicate that cell density is much higher in epicardium than endocardium, which may cause a fallacious impression of higher transcript intensity in the compact myocardium. Quantitative PCR confirmed qualitative findings of the in situ hybridization. omERG transcript expression was higher in atrium than ventricle, and higher in CA than WA trout (P < 0.05). Although statistically significant, the difference in transcript level between acclimation groups was not as large as that of the current.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Effects of Temperature Acclimation on Electrophysiology of IKr
The whole cell conductance of fully activated IKr was almost the same in CA trout at 4°C and in WA trout at 18°C, indicating a complete thermal compensation of the IKr in trout atrial myocytes. Function and number of ERG channels can be regulated by multiple mechanisms (see introduction), and, in principle, any of those could be involved in positive thermal compensation of the trout cardiac IKr. As far as we know, molecular mechanisms of thermal compensation of the IKr have not been previously examined in fish or any other ectothermic vertebrate.
Temperature dependence (Q10) of the whole cell IKr conductance was slightly over 1.3 between 4 and 18°C, which is close to the thermal dependence of diffusion and similar to the Q10 value of the mammalian IKr (42, 33). The constancy of Q10 over the whole temperature range and linearity of the whole cell conductance indicate that we were measuring diffusion of K+ ions through fully open channels without interference of gating kinetics, which has much stronger temperature dependence. Similar whole cell conductance of the IKr in CA trout at 4°C and in WA trout at 18°C strongly suggests that the number of functional omERG channels must be substantially higher in CA than WA rainbow trout. At 11°C, IKr conductance was about twice as large in CA as WA trout, suggesting that the number of omERG channels would be approximately doubled by CA.
ERG channels have complex kinetics characterized by relatively slow activation and very rapid inactivation, which results in inward rectification at positive membrane potentials (26). As an outcome of its kinetic properties, IKr is strongly repolarizing at the late plateau phase of cardiac AP. Examination of whole cell kinetics of the IKr clearly demonstrates that kinetic properties of the trout atrial IKr are not modified by thermal acclimation. Kinetics of activation, deactivation, and inactivation were practically indistinguishable in CA and WA trout when measured at 11°C. In the absence of thermal compensation, gating of the omERG will be strongly affected by acute temperature changes. It should be noted, however, that activation kinetics of the IKr was only slightly slowed down between 11 and 4°C, suggesting trout cardiac ERG channels retain their activity fairly well at low temperatures.
Acute temperature effect was also evident in the voltage dependence of steady-state inactivation, which, in the WA trout, was shifted 15 mV in hyperpolarizing direction by temperature change from 18 to 11°C. However, no further shift was observed between 11 and 4°C in the CA trout, again demonstrating thermal resistance of trout cardiac IKr at low temperatures. In mammalian human ERG (hERG) channels, acute temperature decrease shifts activation and inactivation curves in opposite directions in the voltage axis, thereby causing a strong reduction of the window IKr (33). Because of the thermal resistance of steady-state activation (between 4 and 18°C) and inactivation (between 11 and 4°C), temperature sensitivity of steady-state occupancy of the open state is less in rainbow trout than in humans. Collectively, electrophysiological experiments indicate a complete thermal compensation of the trout atrial IKr and strongly suggest that this is achieved by doubling of the number of active ERG channels, which may be further aided by thermal resistance of steady-state activation and inactivation at temperatures <11°C.
Abundance of omERG mRNA was examined to resolve whether transcript expression could explain temperature-induced and regional differences of the trout IKr. In situ hybridization showed that omERG is uniformly expressed in atrial wall, whereas, in ventricle, omERG expression seems to be stronger in epicardial than endocardiac myocardium. Transmural differences in ERG channel expression have been previously reported for ferret ventricle and are assumed to be physiologically important (10). However, in rainbow trout ventricle, the transmural gradient of omERG staining intensity could be caused by higher myocyte density in epicardium compared with endocardium and, therefore, may not be real. Careful electrophysiological and molecular comparisons of spongious and compact myocardium of the trout ventricle are needed to resolve putative transmural differences in IKr and their physiological significance. Clearly, transcriptional regulation or transcript stability seems to be responsible, at least partly, for spatial and temperature-related differences in IKr phenotype of the trout heart.
In some respects, temperature dependence of the trout cardiac IKr seems to differ from that of the mammalian heart (see Effects of Temperature Acclimation on Electrophysiology of IKr above), suggesting differences either in ERG channel structure or in the regulation of channel activity between ectothermic and endothermic vertebrates. Although the deduced amino acid sequence of the cloned omERG gene shows high-sequence similarity with ERG1 proteins of other vertebrates, omERG sequence differs in several positions from the ERG of endothermic animals. Intracellular NH2- and COOH termini of the omERG have lower similarity with mammalian ERGs, and especially the sequence and length of the COOH terminus of the omERG varied considerably. Noticeably, COOH terminus of the omERG is shorter than in mammalian channels. Pre-mRNA processing is considered to be important in generating molecular diversity in vertebrates (for a review, see Ref. 27) and, in principle, could produce functionally different COOH-terminal isoforms of the omERG channels. However, we could not find any acclimation-induced differences in COOH terminus of the omERG, suggesting that pre-mRNA processing does not contribute to the temperature-related properties of the trout IKr current. The short COOH terminus of the omERG probably does not prevent formation of functional channels, as alternatively spliced isoform of the human heart, hERGUSO, is able to form functional channel, despite its truncated COOH terminus (6, 18). However, this needs to be verified by expression of the omERG and electrophysiological characterization of the IKr in a heterologous system. Zebrafish ERG protein, which is longer than omERG, generates typical IKr currents in Xenopus oocytes (5). Another interesting structural difference between mammalian and fish ERG is the slightly longer S5-P linker of the fish ERG, which lines the outer mouth of the pore and is critical for fast inactivation and K+ selectivity of the ERG channel (21). It is surprising that functionally important region shows so marked sequence and size differences between mammalian and fish channels. It should be noted, however, that the S5-P linker is quite similar in rainbow trout and zebrafish, indicating that the structure of the S5-P linker is probably conserved among fish species.
Glycosylation regulates trafficking of ERG proteins within the cell. In the ER, newly synthesized ERG proteins are modified by asparagine (N)-linked glycosylation to form core glycosylated ERG proteins, which are transported to the Golgi apparatus for further glycosylation before their incorporation in the plasma membrane (for review, see Ref. 30). The two consensus sites of the N-linked glycosylation in the S5-S6 linker are similar in mammalian and fish ERGs, suggesting a similar glycosylation process. Trafficking process of ERG channels also provides means to regulate the number of functional ERG channels, independent from the transcriptional regulation. Reduced temperature improves ER and Golgi processing and surface membrane expression of the transport deficient mutants of the hERG in cell culture conditions (4, 41). In the cold, immature ERG proteins stay longer in the ER, giving chaperones more time to guide correct folding, which reduces misfolding and thus degradation of incorrectly folded ERG channels (13). Contribution of this mechanism to the number of sarcolemmal omERG channels remains to be shown. In addition to the glycosylation, the ER retention signal (R-G-R) in the COOH-terminal tail of the mammalian ERG is involved in ERG trafficking (19). It prevents transport of the channel to the plasma membrane, if it is not masked and inactivated by the COOH terminus of the protein (19). However, both omERG (this study) and zebrafish ERG (39) lack the ER retention signal, suggesting that this inhibitory step is absent in the trafficking of fish ERG channels.
Protein kinases A (PKA), B (PKB), and C (PKC) are all known to regulate hERG channel activity via different signaling pathways. PKA-induced phosphorylation through cAMP cascade decreases whole cell hERG current by a positive voltage shift of the activation curve (31), whereas basal activation of PKB maintains hERG channel function via a phosphoinositide 3-kinase-dependent and -independent pathways (40). Stimulation of -adrenergic receptors causes hydrolysis of PIP2 and reduces ERG current via PKC- and PKA-dependent signaling pathways. PIP2 interacts with a polycationic region of the hERG COOH terminus and increases IKr by negative voltage shift of the steady-state activation (9). PKA, PKB, and PKC are all serine/threonine protein kinases for which mammalian ERG channels contain several putative consensus sequence motifs. Several putative phosphorylation sites also exist in the omERG channel, but the majority of them are different from the mammalian phosphorylation sites. For example, the polycationic sequence of the omERG, involved in PIP2 signaling, has a higher concentration of positively charged amino acids than human ERG, which is likely to cause stronger attraction of PIP2 to its functional site. The protein kinase-dependent regulation of omERG is potentially very important for thermal acclimation, since all serine/threonine kinases can be modulated through adrenergic receptors, and adrenergic sensitivity of the trout heart is increased by CA (17). Clearly, physiological significance of these sequence differences between fish and mammalian ERG channels for different functional demands of the IKr in ectothermic and endothermic hearts earns further studies.
Conclusions
In rainbow trout atrial myocytes, CA increases whole cell conductance of the IKr 1.91-fold, suggesting that the number of functional omERG channels is almost doubled by CA. Increased number of functional channels, together with thermal resistance of voltage dependence of steady-state activation and inactivation and insensitivity of activation kinetics at low temperatures, provides almost complete thermal compensation of the IKr. omERG channels are present in atrial and ventricular myocytes and in sinoatrial pacemaker and the sinoatrial valve. Expression of omERG transcripts is higher in atrium than ventricle, and in both chambers higher in CA than WA fish. Cold-induced increase in IKr is probably vital in restraining temperature-dependent prolongation of cardiac AP in the cold, thereby enabling compensatory changes in heart rate and refractoriness of electrical and mechanical activity of the heart.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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), PKA-mediated phosphorylation of mammalian (
), and omERG channels (
) are indicated. ER retention signal (***) and cell attachment signal (xxx) are indicated. B: cylinders indicate transmembrane 