Finol-Urdaneta RK et al. (2006), Molecular and Functional Differences between He...

XB-ART-34503

J Gen Physiol 2006 Jul 01;1281:133-45. doi: 10.1085/jgp.200609498.

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Molecular and Functional Differences between Heart mKv1.7 Channel Isoforms.

Finol-Urdaneta RK , Strüver N , Terlau H .

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Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

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Species referenced: Xenopus laevis
Genes referenced: kcna7 tbx2 tff3.7

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	Figure 1. mKv1.7 nucleotide and amino acid sequence. (A) Sequence alignment of the 5′ region of Kcna7. Nucleotide and amino acid sequence corresponding to the 5′ UTR and coding sequence for the cytoplasmic N-terminal domain of the mKv1.7 channels. The mouse chromosome 7 sequence AC073711, mouse cDNA fragment AJ409348 (Kashuba et al., 2001), and accession no. AY779767 (this work) sequences are >99% identical. Stop codon is marked with an asterisk, and initiation of the translation sites are underlined. Amino acids in bold correspond to the putative mKv1.7 N-terminal domain. (B and C) Schematic representation of the mKv1.7L and mKv1.7S constructs including the six hydrophobic domains (S1–S6), N and C cytoplasmic termini, and the pore region (H5). Initiation methionines are indicated by circles. The T1 domain is indicated by gray shaded boxes.
	Figure 2. Activation of mKv1.7 channels. Representative outward currents in response to 500-ms depolarizing test potentials ranging from −60 to +80 mV in 10-mV increments, extracellular solution NFR, Vh= −100 mV. mKv1.7L is shown in black (A) and mKv1.7S in gray (B). (C) Steady-state activation curves for mKv1.7L (n = 20, black) and mKv1.7S (n = 15, gray). Mean ± SEM of the relative conductances are plotted against the potential. The smooth line corresponds to Boltzmann fits to the data. (D) Rise time plot of mKv1.7L (n = 13) and mKv1.7S (n = 20) currents measured as the time in ms required for reaching 75% of Imax (RT75) from on-cell patch clamp experiments. mKv1.7L data are shown in black and mKv1.7S in gray. The inset in D presents superimposed early current traces of on-cell patch clamp recordings of mKv1.7L and mKv1.7S channels.
	Figure 3. Inactivation of mKv1.7 channels. (A) Normalized and superimposed mKv1.7L (black) and mKv1.7S (gray) TEVC current traces elicited by a 2.5-s pulse from −60 to +80 mV, in 20-mV steps. (B) Bar diagram of the fractional current (steady-state current divided by peak current amplitude, Iss/Ipeak) of Kv1.7L and Kv1.7S channels (n = 15 and n = 13, respectively). (C) Inactivation time constants derived from monoexponential fits to the inactivation process, mKv1.7L (n = 15) and mKv1.7S (n = 13). (D) Steady-state inactivation was estimated by measuring the peak current amplitude elicited by 30-ms test pulses to 40 mV after 1.5-s prepulses to a −120 to 20 mV potential range. Current is plotted as a fraction of the maximum peak current and fitted with a Boltzmann function (n = 6 and 5 for mKv1.7L and mKv1.7S, respectively). (E) Recovery from inactivation. Time course of the recovery from inactivation of mKv1.7L and Kv1.7S channels is shown in the inset. The mean ± SEM P2/P1 ratio is shown as a function of time (2.5K+o mKv1.7L: black, n = 15; 2.5K+o mKv1.7S: gray, n = 6; 117.5 K+o mKv1.7L: red, n = 4).
	Figure 4. Cumulative inactivation of mKv1.7 channels, [K+]o dependence. Currents were elicited by a train of depolarizing pulses with duration of 5 s to +40 mV with an interpulse interval of 1 s from a Vh of −100 mV (inset in C). (A and B) Eight consecutive current traces from mKv1.7L and mKv1.7S, respectively. The insets contain first 500 ms of the corresponding currents of mKv1.7L in black and mKv1.7S in gray. (C) Cumulative inactivation time course plotted as the fraction of the current left after each consecutive pulse (Inth pulse/I1st pulse vs. pulse number). Smooth lines correspond to monoexponential fits (mKv1.7L in black, n = 15; and mKv1.7S in gray, n = 12). (D and E) Effect of different potassium concentrations over cumulative inactivation of mKv1.7L and mKv1.7S, respectively. Data are given as average values of four oocytes per K+ concentration (K+o in mM: ◯, 2.5; ▵, 5; ▪, 20; ▴, 50; ♦, 117.5).
	Figure 5. Redox sensitivity of mKv1.7. (A) Superimposed on-cell and inside-out patch clamp records from the same patch containing mKv1.7L channels. Gray corresponds to recordings in the on-cell configuration; black represents the currents under inside-out condition. Test pulses between −60 and +80 mV in 20-mV steps; Vh = −100 mV. (B) Normalized TEVC current traces from mKv1.7L exposed to different redox conditions (black corresponds to NFR; green, DTT; red, DTDP; and gray is washout with NFR). The inset presents the nonnormalized experiment. The dashed lines correspond to 0 current level. (C) mKv1.7S from a patch experiment (colors as in A). (D) mKv1.7S TEVC experiment (colors as in B). (E) Relative change in the inactivation time constant upon oxidation of Kv1.7L (solid bars, n = 6) and Kv1.7S (empty bars, n = 4) channels. Time constants obtained from monoexponential fits to the current decay upon +40-mV pulses under control conditions (τcontrol) divided by inactivation time constant under oxidizing conditions (τoxidized, τinside out, τDTDP).
	Figure 6. N-terminal determinants of mKv1.7 channels redox sensitivity. (A) Scaled and superimposed current traces of the different N terminus mutants (+40 mV, 500 ms, Vh = −100 mV) of Kv1.7L. Black traces correspond to recordings under control conditions (NFR) and red traces are currents recorded in the presence of 300 μM DTDP. The dashed lines correspond to 0 current. Mutations are indicated in each panel. (B) Relative change in the inactivation time constant upon oxidation of mKv1.7 mutant channels. Time constants obtained from monoexponential fits to the current decay upon +40 mV pulses under control conditions (τcontrol) divided by inactivation time constant under DTDP oxidizing conditions (τoxidized) (mean ± SEM; n = 5–15).

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