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. 2009 May;12(5):611-7.
doi: 10.1038/nn.2291. Epub 2009 Mar 29.

Oxidation of a potassium channel causes progressive sensory function loss during aging

Shi-Qing Cai  1 , Federico Sesti
Affiliations

Oxidation of a potassium channel causes progressive sensory function loss during aging

Shi-Qing Cai et al. Nat Neurosci. 2009 May.

Abstract

Potassium channels are key regulators of neuronal excitability. Here we show that oxidation of the K(+) channel KVS-1 during aging causes sensory function loss in Caenorhabditis elegans and that protection of this channel from oxidation preserves neuronal function. Chemotaxis, a function controlled by KVS-1, was significantly impaired in worms exposed to oxidizing agents, but only moderately affected in worms harboring an oxidation-resistant KVS-1 mutant (C113S). In aging C113S transgenic worms, the effects of free radical accumulation were significantly attenuated compared to those in wild type. Electrophysiological analyses showed that both reactive oxygen species (ROS) accumulation during aging and acute exposure to oxidizing agents acted primarily to alter the excitability of the neurons that mediate chemotaxis. Together, these findings establish a pivotal role for ROS-mediated oxidation of voltage-gated K(+) channels in sensorial decline during aging in invertebrates.

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Figures

Fig. 1
Fig. 1. KVS-1 channels expressed in mammalian cells are susceptible to redox modulation
A Representative macroscopic KVS-1 currents elicited by voltage jumps from -80 mV to +120 mV in 20 mV increments (inset) in control and after application of 0.3 mM CHT for five minutes in the test solution, washout and application of 0.3 mM DTT for 5-10 minutes in the test solution. B Representative ΔNIRD currents in control and after application of 1 mM H2O2 for 2-3 minutes, washout, and application of 0.3 mM DTT for 5-10 minutes.
Fig. 2
Fig. 2. Cysteine 113 mediates redox modulation of KVS-1
A Representative macroscopic C113S KVS-1 currents elicited by voltage jumps from -80 mV to +120 mV in 20 mV increments in control and after application of 0.3 mM CHT for 5 minutes in the test solution, washout and application of 0.3 mM DTT for 5-10 minutes in the test solution. B Representative ΔNIRD-C95S currents in control and after application of 1 mM H2O2 for 2-3 minutes, washout, and application of 0.3 mM DTT for 5-10 minutes.
Fig. 3
Fig. 3. Protected chemosensory function in C113S worms
A Chemotaxis to biotin in N2 (parental control strain), tm2034 (kvs-1 null), wild type-KVS-1 (WT) and C113S-KVS-1 (C113S), L4 worms. The chemotaxis-defective eat-4(ky5), which harbors a mutation in a vesicular glutamate transporter necessary for glutamatergic neurotransmission in C.elegans, was employed as “sensory-defective” positive control . n = 4 experiments. B Chemotaxis to biotin in control conditions (black) and in worms exposed to hydrogen peroxide (light grey). Young adult worms were soaked in M9 buffer containing 1 mM H2O2 (for 20 minutes) or M9 buffer (control), allowed to recover for 30 minutes , transferred to a test plate and tested for chemotaxis. n = 5 experiments for N2, wild type-KVS-1 and C113S-KVS-1 and n = 3 experiments for kvs-1 KO. C As in B for worms exposed to 0.5 mM CHT for 40 minutes. n = 5 experiments. D As in B for Pflp-6::wild type-KVS-1 and Pflp-6::C113S-KVS-1 worms. n = 4 experiments. E Forward movement phenotype in the indicated genotypes in control (black) and after exposure to 1 mM H2O2 (light grey). n ≥ 11 animals/bar. F Mean average speeds in the indicated genotypes in control (black) and after exposure to 1 mM H2O2 (light grey). n ≥ 10 animals/bar. Data are presented as mean ± standard error of the mean (s.e.m). Statistically significant differences are indicated with * (0.01 < P < 0.05) and ** (P < 0.01).
Fig. 4
Fig. 4. Chemosensory loss is lessened in C113S worms during ageing
A Chemotaxis to biotin in the indicated genotypes (N2 black, wild type-KVS-1 light grey, C113S-KVS-1 white, age-1(hx546) dark grey) at the indicated time points. T= 20 °C. An experiment started with 600-1000 age-synchronized worms/genotype that were scored for chemotaxis at the indicated time points (∼100 worms/time point). N2, wild type-KVS-1 and C113S-KVS-1, n = 8 experiments. Age-1(hx546), n = 3 experiments. B Chemotaxis to biotin in control conditions (black) and in worms exposed to DTT (light grey). Twelve days old worms were soaked in M9 buffer + 1 mM DTT orM9 buffer (control) for 30 minutes, allowed to recover for 1-2 hours, transferred to a test plate and tested for chemotaxis. n=3 experiments. C Chemotaxis to biotin in four days (black) and 12 days (light grey) old Pflp-6::wild type-KVS-1 and Pflp-6::C113S-KVS-1 worms. n = 3 experiments. D Forward movement phenotype in the indicated genotypes in 12 days old worms. n ≥ 11 animals/bar. E Mean average speed in the indicated genotypes in 12 days old worms. n ≥ 10 animals/bar. F SOD activity in 4 (black) and 12 days old (light grey) worms. SOD activity was normalized to the total protein content of the lysate. n = 3 experiments. Data are presented as mean ± standard error of the mean (s.e.m). Statistically significant differences are indicated with * (0.01 < P < 0.05) and ** (P < 0.01).
Fig. 5
Fig. 5. KVS-1 conducts the A-type current in ASER neurons
A Representative whole-cell currents elicited by voltage jumps from -80 mV to +80 mV (inset) in N2, wild type-KVS-1, C113S-KVS-1 and tm2034 ASER neurons. Currents were recorded four days after seeding. B Inactivation rates in N2 (hollow squares), wild type-KVS-1 (squares) and C113S-KVS-1 (hollow circles) currents. Time constants were calculated by fitting macroscopic currents to a single exponential function (eqn. S-1). n= 38, 13 and 25 cells for respectively N2, wild type-KVS-1 and C113S-KVS-1. C Peak current-voltage relationships in ASER neurons of N2, wild type-KVS-1, C113S-KVS-1 worms and steady-state current-voltage relationship in ASER neurons of tm2034 worms (triangles). n = 38, 13, 25 and 23 cells for respectively N2, wild type-KVS-1, C113S-KVS-1 and tm2034. ASER neurons were marked by the Pgcy-5::gfp reported which specifically expresses in this neuron type . Data are presented as mean ± standard error of the mean (s.e.m).
Fig. 6
Fig. 6. Native KVS-1 currents are modified by oxidizing agents
A Representative whole-cell currents evoked in a four days old N2 ASER neuron by single voltage jumps from -80 mV to +80 mV (inset) before and after application of 1 mM H2O2 in the bath solution. Right, fractional current, Isteady/Ibeginning, at +80 mV before and after exposure to 1 mM H2O2, n = 3 cells. B Fractional currents at +80 mV in four days old N2, wild type-KVS-1 and C113S-KVS-1 neurons in the absence (black, n = 15, 14 and 11 cells, respectively) and presence of 0.25 mM H2O2 (light grey, n = 12, 11 and 9 cells respectively) or 0.25 mM CHT (white, n = 10, 11 and 5 cells respectively) in the patch pipette. Data are presented as mean ± standard error of the mean (s.e.m). Statistically significant differences are indicated with * (0.01 < P < 0.05) and ** (P < 0.01).
Fig. 7
Fig. 7. Native KVS-1 currents are modified by endogenous ROS
A Representative whole-cell currents elicited by single voltage jumps from -80 mV to +80 mV (inset) in a 12 days old N2, wild type-KVS-1, C113S-KVS-1 and age-1(hx546) ASER neuron. Inset, representative steady-state current-voltage relationships in 12 days N2 (squares) and C113S-KVS-1 (circles) cells. B Mean fractional current at +80 mV, in 4 days (black) and 12 days (light grey) old neurons in the N2, wild type-KVS-1, C113S-KVS-1 and age-1(hx546) genotypes. Number of cells, are, n = 38, 13, 25 and 15 respectively at day 4 and n = 29, 24, 23 and 15 cells respectively at day 12. C Representative whole-cell currents evoked in an N2 ASER neuron by single voltage jumps from -80 mV to +80 mV (inset) before and after application of 0.3 mM DTT in the bath solution. Right, fractional current at +80 mV before and after exposure to DTT, n = 3 cells. D Mean fractional current in 12 days old N2 or wild type-KVS-1 neurons recorded in the absence (black, n = 21 and 19 cells, respectively) or presence of 0.2 mM DTT in the pipette solution (light grey, n = 12 and 13 cells, respectively). E Representative potentials evoked in a 4 days old N2 ASER cultured neuron in response to 0.5 s current injections from -4 pA to 20 pA in 4 pA increments (current protocol in the inset of Fig. 7 f). Inset, whole-cell currents recorded in the same cell evoked by 1 s voltage steps from -80 mV to +80 mV in 20 mV increments. F As in (E) in a 12 days old N2 ASER neuron. G Steady-state voltage-current relationships in 4 days old N2 neurons (filled squares, fractional current = 0.49 ± 0.08, n = 8 cells), 12 days old N2 neurons (hollow squares, fractional current 0.98 ± 0.07, n = 7 cells), 4 days old C113S-KVS-1 neurons (filled circles, fractional current 0.50 ± 0.04, n = 6 cells) and 12 days old C113S-KVS-1 neurons (hollow circles, fractional current 0.43 ± 0.04, n = 7 cells). Data are presented as mean ± standard error of the mean (s.e.m). Statistically significant differences are indicated with * (0.01 < P < 0.05) and ** (P < 0.01).
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