Jackson HA et al. (2012), Asymmetric divergence in structure and function...

XB-ART-47074

PLoS One 2012 Jan 01;711:e47590. doi: 10.1371/journal.pone.0047590.

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Asymmetric divergence in structure and function of HCN channel duplicates in Ciona intestinalis.

Jackson HA , Hegle A , Nazzari H , Jegla T , Accili EA .

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Hyperpolarization-activated Cyclic Nucleotide (HCN) channels are voltage-gated cation channels and are critical for regulation of membrane potential in electrically active cells. To understand the evolution of these channels at the molecular level, we cloned and examined two of three HCN homologs of the urochordate Ciona intestinalis (ciHCNa and ciHCNb). ciHCNa is like mammalian HCNs in that it possesses similar electrical function and undergoes N-glycosylation of a sequon near the pore. ciHCNb lacks the pore-associated N-glycosylation sequon and is predictably not N-glycosylated, and it also has an unusual gating phenotype in which the channel's voltage-sensitive gate appears to close incompletely. Together with previous findings, the data support an evolutionary trajectory in which an HCN ancestor underwent lineage-specific duplication in Ciona, to yield one HCN with most features that are conserved with the mammalian HCNs and another HCN that has been uniquely altered.

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Species referenced: Xenopus laevis
Genes referenced: actl6a camp hcn1 hcn2 hcn3 hcn4 mapt uqcc6

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	Figure 2. Phylogenetic pattern and N-linked glycosylation of a subset of Ciona HCNs.A. An alignment of the pore region produced by ClustalX and generated by GeneDoc. Red boxes indicates known and putative HCN N-linked glycosylation site and the yellow line indicates the division between the presence and absence of this functional site. B. An HCN cladogram generated by aligning the sequences shown as described in the text. “X” indicates the predicted emergence point of N-linked glycosylation in the HCN family. Sequences other than those in Figure 1, and other than those for Oikopleura dioica and Branchiostoma floridae (lancelet), are: gi: 260829977; Strongylocentrotus purpuratus (sea urchin) gi:47551101; Panulirus interruptus, splice variant I (lobster) gi:115334851; Apis mellifera (bee) gi:33355927; Drosophila melanogaster, isoform B, gi:84795752; Aedes aegypti (mosquito) gi:108875949. C. Western blot of membrane fractions from oocytes injected with cRNA of ciHCNa, ciHCNa-N380Q or ciHCNb, untreated (−) or treated (+) with PNGaseF and probed with anti-V5 antibody. Actin, robed with an anti-actin antibody, was used as a loading control. ciHCNa, but not ciHCNa-N380Q or ciHCNb, is shifted to a lower molecular mass in the presence of PNGaseF. Molecular weights, indicated by the short black bars to the right of the blots, are 130 kDa (top), 100 kDa (upper middle) and 70 kDa (lower middle) and 35 kDa (bottom). D. Western blot of whole cell lysates from CHO cells transfected with 1.5 ug cDNA of ciHCNa, ciHCNa-N380Q or ciHCNb, either treated (+) or untreated (−) with PNGaseF and probed for using an anti-V5 antibody. Molecular weights, indicated by the short black bars to the right of the blots, are 95 kDa (top), 72 kDa (middle) and 34 kDa (bottom). Predicted mass of Ciona HCN channels without post-translational modification, is 78.8 kDa and 93.3 kDa for ciHCNa and ciHCNb, respectively.
	Figure 3. Variable opening by hyperpolarization and cesium block of current produced by the two Ciona HCNs.A. Black current traces elicited by a single 6 second hyperpolarizing pulse to −70 mV, from a holding potential of −10 mV, followed by pulse back to −30 mV, in an oocyte containing ciHCNa (above) or ciHCNb (below), bathed an extracellular solution containing 96 mM potassium. The voltage protocol utilized is shown between the two sets of current traces. The black vertical lines to the left indicate the instantaneous (Iinst) and the slowly-activating (Ih) components of the current trace. Gray current traces were produced by the same voltage protocol, following incubation with the same extracellular solution, with the addition of 5 mM Cs+, for three minutes while clamped at −10 mV. The dotted lines represent the zero current level. Capacitive transients elicited upon current activation have been removed for clarity. B. Plots of amplitudes of Iinst and Ih before and during perfusion with Cs+, as indicated. The number of oocytes that were used in each group are shown above each bar in brackets. Values represent mean ± s.e.m.; those in black and gray represent with and without Cs+. *indicates a significant difference induced by Cs+ (p<0.05 in a two-tailed paired t-test).
	Figure 4. Potassium passes through both Ciona HCNs and enhances current flowing through them.A. Current traces elicited by a multi-step protocol in oocytes expressing either Ciona HCN isoform to obtain instantaneous Ih versus voltage relationship, carried out in low and high concentrations of extracellular potassium. The voltage protocol is shown below the current traces. The first set of voltage pulses allow for the determination of instantaneous current at each test voltage; the second set of pulses allows for the determination of total instantaneous current at each test voltage, following Ih activation at a potential at which the current is close to fully activated. Subtracting the latter from the former yields the total instantaneous Ih at each test voltage. B. Plots of instantaneous Ih amplitude versus test voltage at low and high concentrations of extracellular potassium as determined in ‘A’. Values represent mean ± s.e.m. at each test voltage; light gray/squares for low 5 mM K+, dark gray/circles for 10 mM K+, and (black/triangles) for 96 mM K+. C. Bar graphs showing the reversal potentials and slope conductance for each ciona HCN in different concentrations of potassium; these were determined from experiments in ‘A’ where values from individual experiments were fit by a straight line through the linear portion of the curve across the x-axis. The number of oocytes that were used in each group is shown above each bar in brackets; reversal potential and slope conductance were obtained from the same current data. Values represent mean ± s.e.m. for each potassium concentration. Statistical significance was determined using a one-way ANOVA and post-tukey multiple comparison test. * indicates difference between 96 mM potassium (p<0.05) and 5 mM and # indicates a difference between 96 mM potassium and 10 mM potassium (p<0.05). Light gray, dark gray and black bars represent values for 5, 10 and 96 mM potassium.
	Figure 5. Sodium passes through both Ciona HCNs but does not enhance current flowing through them.A. Plots of instantaneous Ih versus voltage at the indicated concentrations of extracellular cations. Values were determined from current traces elicited using the same protocol as for Fig. 5 and represent mean ± s.e.m. for each test voltage; black line/black squares for 100 mM K+, light gray line/black circles for 80 mM Na+/20 mM K+, black line/black upward triangles for 80 mM Li/20 mM K+, black line/black downward triangles for 20 mM K+. Lines represent linear fits through the mean values. B. Bar graphs showing the reversal potentials and slope conductance for each Ciona HCN, determined from the plots in ‘B’ where values from individual experiments were fit by a straight line through the linear portion of the curve across the x-axis. The number of oocytes that were used in each group is shown below each bar in brackets; reversal potential and slope conductance were obtained from the same current voltage data. Values represent mean ± s.e.m. for solution used, which were compared using a one-way ANOVA and tukey's post test. * indicates a difference (p<0.05) when compared to all other conditions. ** indicates a difference (p<0.05) when compared to * or *. * indicates a difference (p<0.05) when compared to ** or *.
	Figure 6. Opening of ciHCNs is facilitated by a rise in intracellular cAMP.A. Current traces for ciHCNa (above) and ciHCNb (below), elicited by the staggered voltage protocols shown above each set of current traces. “Tail” currents were elicited at −30 mV for ciHCNa and +30 mV for ciHCNb, and an example for each isoform is highlighted by a solid black arrow. B. Mean activation curves determined from single order Boltzmann fits of plots of normalized tail currents versus voltage from individual experiments and oocytes [35], under different conditions. The gray and black lines represent the mean curves obtained from control experiments and from experiments carried out in the presence of 10 mM 8-bromo cAMP, respectively. Number of experiments (representing one oocyte per experiment), without and with cAMP, were 14 and 11, respectively, for ciHCNa, and 8 and 8, respectively, for ciHCNb. C. Plot of mean V½ values ± s.e.m. determined by fitting the tail current data, obtained from individual experiments in oocytes expressing ciHCNa or ciHCNb in the absence (open squares) or presence of 8-Br-cAMP (filled squares), with a Boltzmann equation. *indicates significant difference (p<0.05, two-tailed unpaired t-test) between values obtained in control solution as compared to those obtained in a solution containing 8-Br-cAMP. The numbers of oocytes used per group are as for ‘B’.
	Figure 7. The time course of Ih activation and deactivation is different between ciHCNa and ciHCNb.A. (Left) Sample current traces elicited by a hyperpolarization of the membrane potential from a holding potential of −10 mV to −90 (ciHCNa) or −70 mV (ciHCNb). The slow portion of the current traces obtained by hyperpolarization to −90 or −70 mV (Ih) was fit with a double (ciHCNa) or single (ciHCNb) exponential function. The sample fits of these traces are shown in dark gray, along with a plot of the residuals of the fits in light gray at the top of each current trace. (Right) Plots of Tau values, which were obtained from fitting with exponential functions, versus test voltage. B. Plot of the ratio of amplitudes of fast component versus the amplitudes of both the fast and slow component of the double exponential fit for the current traces, obtained from oocytes expressing ciHCNa as described in ‘A’, versus test voltage. For both ‘A’ and ‘B’, the values for “n” refer to the number of oocytes used. For all plots, the values represent the mean ± s.e.m.
	Figure 8. Iinst and Ih flow through the ciHCNb pore in approximately the same proportion.A. Current traces elicited by a hyperpolarization of the membrane potential from a holding potential of −10 mV to −70 mV before (black trace) or after (gray trace) a 15 min incubation with ZD7288. The complete voltage protocol is shown below the current traces. The dotted line represents the zero current level. B. Bar graph of Iinst and Ih before (black bars) and after (gray bars) 15 minute incubation with ZD7288, as carried out in ‘A’. * indicates a significant block of the current component by this agent (p<0.05 in a two-tailed paired t-test). C. Plot of ZD7288-sensitive Iinst versus Ih. Black line represents a linear correlation, which was a significant (r = 0.9, p<0.05). D. Bar graph showing the ratio of Iinst over total current (Iinst+Ih) that is blocked by Cs+ or ZD7288; this ratio represents the proportion of blocked current that is due to block of Iinst. An example of the Cs+ data used for this figure is shown in Figure 2. In ‘B’ and ‘D’, values represent mean ± s.e.m and the numbers in brackets are the numbers of oocytes used. The ZD7288 data in ‘B’, ‘C’ and ‘D’ come from the same 9 oocytes.
	Figure 1. Ciona HCNs show conservation of key regions, but also considerable divergence, with each other and with the mammalian HCN isoforms.Shown is an alignment of ciHCNa and ciHCNb with the four HCN isoforms from the mouse. Identical (black) and conserved (gray) residues among all of the isoforms are shaded. The approximate locations for the six putative transmembrane segments, the pore, C-linker and cyclic nucleotide-binding domain are identified and indicated using solid bars place above the sequence. The sequences shown are: ciHCNa (GI:378407787), ciHCNb (GI:378407789), mouse HCN1 (GI:255760033), mouse HCN2 (GI:6680189), mouse HCN3 (GI:6680191) and mouse HCN4 (GI:124487125). The alignment was carried out using Clustalw (http://bio.lundberg.gu.se/edu/msf2.html) and arranged into the figure by Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).

References [+] :

Altomare, Integrated allosteric model of voltage gating of HCN channels. 2001, Pubmed