Kaku R et al. (2019), Incorporation of one N-glycosylation-deficient ...

XB-ART-56286

Neuroreport 2019 Oct 16;3015:998-1003. doi: 10.1097/WNR.0000000000001310.

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Incorporation of one N-glycosylation-deficient subunit within a tetramer of HCN2 channel is tolerated.

Kaku R , Matsuoka Y , Yang J .

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are glycoproteins N-glycosylated at a specific asparagine residue in the S5-S6 linker region. Previous reports suggested that N-glycosylation-deficient HCN2 N380Q (NQ) channels fail to properly target to the plasma membrane and are unable to form functional ion channels. HCN channels are known to homo- and hetero-oligomerize and it is not known whether HCN2-NQ subunits can oligomerize with wild type (wt) N-glycosylated subunits to form a tetrameric assembly. In the present study, homomeric NQ-mutant resulted in no current, cRNA titration experiments controlling the amount of wt-to-NQ injected into Xenopus oocytes indicated that the observed currents were consistent with a model where presence of a single nonglycosylated subunit in a tetrameric oligomer is tolerated forming functional channels. The activation voltage-dependence described by half-activation voltage and slope factor, and the reversal potential of the wt-NQ oligomeric channels were identical to the wt only tetrameric channels. Further incorporation of the nonglycosylated subunit rendered the channels nonconductive or not incorporated into the plasma membrane.

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Species referenced: Xenopus
Genes referenced: hcn2

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	Fig 1 Two-electrode voltage-clamp records from oocytes injected with wt- or NQ-glycosylation mutant HCN 1 or 2 cRNA. (a) Currents elicited by an activation step protocol from −40 to −140 mV from a holding potential of +10 mV for oocytes injected with the indicated cRNAs. No current was observed on step hyperpolarization for NQ alone or uninjected oocytes. (b) Current magnitude at −120 mV for the various groups of oocytes vs. time after injection. Mean ± SEM from N = 3–12; *P < 0.01 from noninjected control. HCN, hyperpolarization-activated cyclic nucleotide-gated ion channels.
	Fig 2 Current recordings from oocytes injected with variable HCN2 wt-to-NQ ratios. (a) Representative current traces from oocytes injected with all wt cRNA (left) and both wt and NQ-cRNAs at a 1-to-1 ratio. (b) I-V curves from peak currents measured during the activation protocol. wt-to-NQ ratios of 1:0 (closed, n = 7) and 1:1 (open, n = 7). (c) Voltage-dependent activation obtained from the tail current measurements. Symbols as in (b). The fitted traces are normalized Boltzmann distribution given by g/gmax = 1/(1+exp − [(V−V50)/s], where g = conductance, V50 = half-maximal activation voltage, s = slope factor. Please see Table 1 for the numerical values of the fitted data. (d) Channels were activated by a voltage step from −20 to −140 mV and the tail currents measured at variable voltages from −100 to +40 mV (inset). Peak tail currents vs. voltage were plotted from oocytes injected with wt-to-NQ ratios of 1:0 (circles) and 1:1 (triangles) to plot instantaneous I-V curves for estimation of the reversal potential. HCN, hyperpolarization-activated cyclic nucleotide-gated ion channels.
	Fig 3 Peak current magnitudes after injection of variable wt-to-NQ cRNA ratios are consistent with a model where incorporation of one NQ forms functional channels. (a) Theoretical distribution of tetramers consisting of wt (open) and NQ (closed) subunits for variable wt-to-NQ ratios. The curves were calculated from P(x) = n!/(x!(n−x)!) px (1−p)n-1, where P(x) = probability of finding × objects given, n = number of total objects (i.e. 4 for a tetramer), p = probability of wt. (b) Theoretical peak currents (solid lines from left to right) for models assuming only all wt, 3 wt and 1 NQ, 2 wt and 2 NQ, 3 wt and 1 NQ, all NQ tetramers allowing current flow for various wt-to-NQ ratios. Blue circles are experimental data (mean ± SEM) normalized to the current density for 1:0 obtained from oocytes injected with the indicated wt-to-NQ ratios. All currents were recorded at 48–60h after injection. All mean data points are statistically significantly different at P < 0.01 compared to all wt.