XB-ART-46101PLoS One. January 1, 2012; 7 (6): e38205.
Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.
PubMed ID: 22675523
PMC ID: PMC3366920
Article link: PLoS One.
Grant support: R01 GM090029 NIGMS NIH HHS , R01 NS37444 NINDS NIH HHS , P30 HD024064 NICHD NIH HHS , P01 NS037444 NINDS NIH HHS
Genes referenced: dpp6 gapdh kcnd2
Article Images: [+] show captions
|Figure 1. Analysis of DPP6 splice variant expression in CG cells.A) DPP6 gene shows a conserved set of four alternative first exons producing the protein variants DPP6a, DPP6K, DPP6L and DPP6S. B) qRT-PCR using SYBR Green Fluorescence for DPP6 variants from three brain regions: cortex, cerebellum and hippocampus. DPP6a and DPP6K show enhanced expression in cerebellum. C) Normalization controls used to correct for primer amplification efficiency differences. Amplification targets from GAPDH and two DPLP variants were diluted and used to construct amplification curves. Common GAPDH signal was used to ensure consistent dilution of standards. D) Relative expression levels of DPP6 variants in cerebellum following normalization. Due to high levels of expression in CG cells and the abundance of these neurons, these signals essentially report the relative expression of DPP6 variants in CG cells.|
|Figure 2. DPP6K dramatically accelerates inactivation kinetics and leftward shifts steady-state inactivation curve.Xenopus oocytes were injected with cRNAs encoding Kv4.2 and KChIP3a along with either DPP6a, DPP6S, or DPP6K. Transient currents were recorded using two-electrode voltage clamp. (A) Representative normalized current traces generated by voltage steps to +50 mV from a holding potential of −100 mV for 1 sec. Only the first 500 ms are shown. (B) Voltage dependence of steady-state inactivation for the various channel complexes. The steady-state inactivation protocol consisted of a 10-sec prepulse at the indicated potentials and a 250-ms test pulse to +50 mV, with an inter-episode interval of 5 secs. The fraction of available current (I/Imax) was plotted against the prepulse membrane potential. Data are shown as mean ± SEM, and the lines represent fits using Boltzmann functions.|
|Figure 3. DPP6K markedly represses the recovery-accelerating effects of KChIP3a.Representative current traces generated during the two-pulse protocol used to measure recovery from inactivation for Kv4.2+KChIP3a (A), Kv4.2+KChIP3a+DPP6a (B), Kv4.2+KChIP3a+DPP6S (C), and Kv4.2+KChIP3a+DPP6K (D). A 1-sec depolarization to +50 mV was delivered to maximally inactivate the channels, followed by an increasing recovery interval at −100 mV before applying a 250-ms test pulse at +50 mV to check the degree of recovery from inactivation. (E) Fractional recovery was plotted as a function of the interval duration at −100 mV. The residual value at the end the first pulse was subtracted from the peak current values of the first and second pulses, and the fractional recovery was determined by dividing the peak value of the second pulse by that of the first pulse.|
|Figure 4. The DPP6K variable N-terminus is responsible for its distinct functional effects.(A) Voltage dependence of steady-state inactivation of ternary complex containing DPP6K and DPP6K/ΔN16 deletion mutant. The protocol was the same as that of Fig. 2B. (B) The kinetics of recovery from inactivation at −100 mV. The protocol was the same as that of Fig. 3. (C) Alignment of amino acid sequences of DPP6K variable N-terminus from various organisms. Consensus residues are shown in black; conservative substitutions, in gray; non-conservative substitutions, in red. Major evolutionary branch points indicated along the alignment relative to a terminus in placental mammals.|
|Figure 5. Co-assembly of DPP6K and DPP6a in heteromultimeric channel complexes.(A) Outward currents expressed by oocytes co-injected by various combinations of cRNAs, as elicited by depolarization to +40 mV from holding potential of −100 mV. (B) Expected rise and decay of currents if DPP6a and DPP6K subunits do not co-assemble and produce segregated channel populations containing either one alone. (C) Slowing of the time constant of fast inactivation when DPP6a mRNA changes from 100% to 10% mixed with DPP6K mRNA. To get the average value for fast inactivation, the slow phase of inactivation and non-inactivating current were described by exponential fitting and subtracted from the total current. The remaining average fast inactivation time constant was measured by taking the peak current for the fast inactivating fraction divided by its area. The average time constant measured by this method was very similar to the time constant measured by the best single exponential fit to the fast inactivating component. The black and gray lines show the predicted maximal slowing of fast inactivation with four DPP6 and two DPP6 per channel, respectively, with only 1 DPP6a subunit per channel. (D) Recovery from inactivation at −100 mV after a 200 ms-long prepulse (symbols) as compared to predicted results assuming no co-assembly of DPP6a and DPP6K (dashes).|
|Figure 6. Steady-state inactivation of DPP6a∶DPP6K mixed channels.(A) Representative traces for Kv4.2+KChIP3a channels co-expressed with DPP6a alone, DPP6K alone, or with a DPP6a∶DPP6K mixture at 1∶1 or 1∶2 ratios, showing changes in steady-state inactivation at −65 mV. For the colored traces, the channels were held for 30 sec at −65 mV before pulsing to +40 mV for 250 ms to test available current. The black traces show the total currents, from test pulses where the channels were held at −100 mV and experienced no inactivation. (B) Voltage dependence of steady-state inactivation of ternary complexes with homotetrameric and heterotetrameric DPP6 subunits. (C) Progressive shifting of inactivation midpoint with increasing DPP6K ratio. The V0.5i values were plotted against the calculated DPP6K mole fraction. Model assumes independent energetic effects for each DPP6K subunit incorporated into the channel, with symbols measured V0.5i values for summed Boltzmann curves from the model.|
|Figure 7. Reconstitution of native ISA channel from CG cells by heterologous expression in oocytes.(A) Outward transient currents elicited from CG cells and oocytes expressing Kv4.2, a mixture of DPP6a and DPP6K at 1∶2 ratio, and either KChIP3a or KChIP4bL. From a holding potential of −100 mV, either a 200-ms (CG cells) or 1-sec (oocytes) step depolarizations were made from −100 mV to +40 mV at 10 mV increments. (B) Overlapped normalized current traces at +40 mV from the indicated channels. (C) Time constants of inactivation at indicated membrane potentials for ISA from CG cells, Kv4.2+KChIP3a+DPP6a+DPP6K (1∶2), and Kv4.2+KChIP4bL+DPP6a+DPP6K (1∶2). (D) Recovery from inactivation at −100 mV, measured using the two-pulse protocol. (E) Normalized peak conductance-voltage relations (Gp/Gp,max) and steady-state inactivation curves (I/Imax) for ISA from CG cells and reconstituted channel complexes.|