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The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

ABSTRACT: The ? subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming ?(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)? subunit of the human parasite Schistosoma mansoni (?(Sm)), a motif that does not occur in other known Ca(v)? subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, ?(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of ?(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (?(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (?(Sm): ?(fast) = 66 ms; ?(Sm?2-44): ?(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (?(fast) = 42 ms with ?(Sm?2-17); ?(fast) = 40 ms with ?(Sm?2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of ?(Sm) attached to mammalian ?(1b), ?(2a), and ?(3) subunits. For any given mammalian ? subunit, inactivation was faster if it contained the N terminus of ?(Sm) than if it did not. Co-expression of the mammalian ?(2)?-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.

SUBMITTER: Salvador-Recatala V

PROVIDER: S-EPMC2975211 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

Salvador-Recatalà Vicenta V Greenberg Robert M RM

The Journal of biological chemistry 20100907 46

The β subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming α(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)β subunit of the human parasite Schistosoma mansoni (β(Sm)), a motif that does not occur in other known Ca(v)β subunits. When expressed in human embr ...[more]

PMID: 20826800

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Project description:In superior cervical ganglion (SCG) neurons, stimulation of M(1) receptors (M(1)Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M(1)Rs. In addition, techniques that diminish AA's concentration during M(1)R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M(1)R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the beta subunit (Ca(V)beta) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M(1)Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca(V)2.2 and coexpressed with Ca(V)beta1b, Ca(V)beta3, or Ca(V)beta4 but enhanced activity of N channels containing Ca(V)beta2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca(V)beta2a, Ca(V)beta3, and Ca(V)beta4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca(V)beta with Ca(V)2.2. Further experiments with mutated, chimeric Ca(V)beta subunits and free palmitic acid revealed that palmitoylation of Ca(V)beta2a is essential for loss of inhibition. The data presented here fit a model in which Ca(V)beta2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca(V)beta2a toggles M(1)R- or NK-1R-mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M(1)Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca(V)beta2a.

Dataset Information

The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

Publications

The N terminus of a schistosome beta subunit regulates inactivation and current density of a Cav2 channel.

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