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Regulated RNA editing and functional epistasis in Shaker potassium channels.

Ingleby L, Maloney R, Jepson J, Horn R, Reenan R - J. Gen. Physiol. (2009)

Bottom Line: Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells.Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites.Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 2(4) = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

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Kinetics and steady-state properties of inactivation. (A) Time constants of inactivation show that AAGA is distinctly slower than the other isoforms. (B) Diversity of steady-state inactivation curves obtained in response to 100-ms prepulses. (C) Kinetics of recovery from inactivation at −120 mV (see inset for voltage protocol). Theory curves are single exponential relaxations (Table S2).
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fig5: Kinetics and steady-state properties of inactivation. (A) Time constants of inactivation show that AAGA is distinctly slower than the other isoforms. (B) Diversity of steady-state inactivation curves obtained in response to 100-ms prepulses. (C) Kinetics of recovery from inactivation at −120 mV (see inset for voltage protocol). Theory curves are single exponential relaxations (Table S2).

Mentions: We selected 9 of the 16 possible isoforms for detailed functional characterization. Each was expressed transiently in a mammalian cell line, and whole cell currents were characterized. The channel-forming α-subunits were coexpressed with the auxiliary subunit Hyperkinetic, a cytoplasmic protein that associates with Shaker in Drosophila (Chouinard et al., 1995). Because the expression levels were so high in all of these isoforms, we were able to examine whole cell currents carried by Cs+, which is two orders of magnitude less conductive than K+ (Heginbotham and MacKinnon, 1993). Fig. 4 shows examples of families of currents elicited by depolarizations from a holding potential of −120 up to +70 mV. The top row shows unedited (AAAA) and fully edited (GGGG) isoforms. The bottom row shows the two isoforms with the most extreme functional phenotypes in terms of the kinetics of inactivation. GGGA has the fastest, and AAGA the slowest, rates of inactivation among the nine variants we examined. The rate of inactivation during a depolarization is well fit by a single exponential relaxation. Fig. 5 A shows the time constants for these fits. Although most of these kinetic parameters are comparable among the nine isoforms, AAGA stands out as the slowest over a wide range of voltages, with inactivation time constants approximately threefold slower than those of GGGA. Steady-state inactivation also varied among the nine isoforms, with midpoints differing as much as 11.8 mV (Fig. 5 B and Table S2). The fractional extent of inactivation induced by a 100-ms depolarization to −20 mV (Fig. 5 B) also varied among the isoforms, from 0.92 ± 0.02 for GGGA to 0.78 ± 0.01 for AAGA. This steady-state behavior is consistent with the kinetics of inactivation in that the isoform that inactivates most rapidly also inactivates most completely, and the isoform that inactivates most slowly inactivates least completely. Like entry into the inactivated state, the rate of recovery from inactivation at −120 mV, after a 75-ms depolarization to +70 mV (Fig. 5 C, inset), also had an approximately threefold range, with GGGA the slowest and AAGG the fastest (Table S2). Thus, isoforms that inactivate rapidly tend to recover slowly, and vice versa.


Regulated RNA editing and functional epistasis in Shaker potassium channels.

Ingleby L, Maloney R, Jepson J, Horn R, Reenan R - J. Gen. Physiol. (2009)

Kinetics and steady-state properties of inactivation. (A) Time constants of inactivation show that AAGA is distinctly slower than the other isoforms. (B) Diversity of steady-state inactivation curves obtained in response to 100-ms prepulses. (C) Kinetics of recovery from inactivation at −120 mV (see inset for voltage protocol). Theory curves are single exponential relaxations (Table S2).
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fig5: Kinetics and steady-state properties of inactivation. (A) Time constants of inactivation show that AAGA is distinctly slower than the other isoforms. (B) Diversity of steady-state inactivation curves obtained in response to 100-ms prepulses. (C) Kinetics of recovery from inactivation at −120 mV (see inset for voltage protocol). Theory curves are single exponential relaxations (Table S2).
Mentions: We selected 9 of the 16 possible isoforms for detailed functional characterization. Each was expressed transiently in a mammalian cell line, and whole cell currents were characterized. The channel-forming α-subunits were coexpressed with the auxiliary subunit Hyperkinetic, a cytoplasmic protein that associates with Shaker in Drosophila (Chouinard et al., 1995). Because the expression levels were so high in all of these isoforms, we were able to examine whole cell currents carried by Cs+, which is two orders of magnitude less conductive than K+ (Heginbotham and MacKinnon, 1993). Fig. 4 shows examples of families of currents elicited by depolarizations from a holding potential of −120 up to +70 mV. The top row shows unedited (AAAA) and fully edited (GGGG) isoforms. The bottom row shows the two isoforms with the most extreme functional phenotypes in terms of the kinetics of inactivation. GGGA has the fastest, and AAGA the slowest, rates of inactivation among the nine variants we examined. The rate of inactivation during a depolarization is well fit by a single exponential relaxation. Fig. 5 A shows the time constants for these fits. Although most of these kinetic parameters are comparable among the nine isoforms, AAGA stands out as the slowest over a wide range of voltages, with inactivation time constants approximately threefold slower than those of GGGA. Steady-state inactivation also varied among the nine isoforms, with midpoints differing as much as 11.8 mV (Fig. 5 B and Table S2). The fractional extent of inactivation induced by a 100-ms depolarization to −20 mV (Fig. 5 B) also varied among the isoforms, from 0.92 ± 0.02 for GGGA to 0.78 ± 0.01 for AAGA. This steady-state behavior is consistent with the kinetics of inactivation in that the isoform that inactivates most rapidly also inactivates most completely, and the isoform that inactivates most slowly inactivates least completely. Like entry into the inactivated state, the rate of recovery from inactivation at −120 mV, after a 75-ms depolarization to +70 mV (Fig. 5 C, inset), also had an approximately threefold range, with GGGA the slowest and AAGG the fastest (Table S2). Thus, isoforms that inactivate rapidly tend to recover slowly, and vice versa.

Bottom Line: Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells.Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites.Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
Regulated point modification by an RNA editing enzyme occurs at four conserved sites in the Drosophila Shaker potassium channel. Single mRNA molecules can potentially represent any of 2(4) = 16 permutations (isoforms) of these natural variants. We generated isoform expression profiles to assess sexually dimorphic, spatial, and temporal differences. Striking tissue-specific expression was seen for particular isoforms. Moreover, isoform distributions showed evidence for coupling (linkage) of editing sites. Genetic manipulations of editing enzyme activity demonstrated that a chief determinant of Shaker editing site choice resides not in the editing enzyme, but rather, in unknown factors intrinsic to cells. Characterizing the biophysical properties of currents in nine isoforms revealed an unprecedented feature, functional epistasis; biophysical phenotypes of isoforms cannot be explained simply by the consequences of individual editing effects at the four sites. Our results unmask allosteric communication across disparate regions of the channel protein and between evolved and regulated amino acid changes introduced by RNA editing.

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