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The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel.

Zeng H, Weiger TM, Fei H, Jaramillo AM, Levitan IB - J. Gen. Physiol. (2005)

Bottom Line: We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD).In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel.These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

ABSTRACT
The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

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Modulation of dSlo by Slob71, Slob65, Slob53, and Slob47. Whole cell currents evoked by depolarizing voltage steps (G) with 110 μM free Ca2+, in cells transfected with vector alone (A), dSlo alone (B), or together with different Slobs as indicated (C–F). Currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents.
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fig3: Modulation of dSlo by Slob71, Slob65, Slob53, and Slob47. Whole cell currents evoked by depolarizing voltage steps (G) with 110 μM free Ca2+, in cells transfected with vector alone (A), dSlo alone (B), or together with different Slobs as indicated (C–F). Currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents.

Mentions: We examined the effects of all the Slobs on dSlo channel function by cotransfecting each with dSlo in CHO cells, which exhibit very low levels of endogenous potassium current (Fig. 3 A). In preliminary experiments, we found that a high concentration of free Ca2+ is required to achieve full dSlo activation and tail current saturation in the presence of Slob57. Therefore we chose to study the modulatory effects of the different Slobs in the presence of 110 μM free Ca2+. Whole cell outward currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents (Fig. 3 G). A comparison of the current traces in the absence (Fig. 3 B) or presence (Fig. 3, C–F) of various Slobs (Slob71, Slob65, Slob53, and Slob47) indicates that there is no change in activation or deactivation kinetics. However there is a small but significant hyperpolarizing shift in the dSlo conductance–voltage relationship in the presence of each of these Slob variants (Fig. 4 and Table I). Such shifts are reminiscent of (although smaller than) those reported previously with HA-tagged Slob57 (Zhou et al., 1999; unpublished data).


The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel.

Zeng H, Weiger TM, Fei H, Jaramillo AM, Levitan IB - J. Gen. Physiol. (2005)

Modulation of dSlo by Slob71, Slob65, Slob53, and Slob47. Whole cell currents evoked by depolarizing voltage steps (G) with 110 μM free Ca2+, in cells transfected with vector alone (A), dSlo alone (B), or together with different Slobs as indicated (C–F). Currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234080&req=5

fig3: Modulation of dSlo by Slob71, Slob65, Slob53, and Slob47. Whole cell currents evoked by depolarizing voltage steps (G) with 110 μM free Ca2+, in cells transfected with vector alone (A), dSlo alone (B), or together with different Slobs as indicated (C–F). Currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents.
Mentions: We examined the effects of all the Slobs on dSlo channel function by cotransfecting each with dSlo in CHO cells, which exhibit very low levels of endogenous potassium current (Fig. 3 A). In preliminary experiments, we found that a high concentration of free Ca2+ is required to achieve full dSlo activation and tail current saturation in the presence of Slob57. Therefore we chose to study the modulatory effects of the different Slobs in the presence of 110 μM free Ca2+. Whole cell outward currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents (Fig. 3 G). A comparison of the current traces in the absence (Fig. 3 B) or presence (Fig. 3, C–F) of various Slobs (Slob71, Slob65, Slob53, and Slob47) indicates that there is no change in activation or deactivation kinetics. However there is a small but significant hyperpolarizing shift in the dSlo conductance–voltage relationship in the presence of each of these Slob variants (Fig. 4 and Table I). Such shifts are reminiscent of (although smaller than) those reported previously with HA-tagged Slob57 (Zhou et al., 1999; unpublished data).

Bottom Line: We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD).In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel.These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

ABSTRACT
The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Show MeSH
Related in: MedlinePlus