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The Journal of Neuroscience, December 24, 2008, 28(52):14176-14188; doi:10.1523/JNEUROSCI.1350-08.2008

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Cellular/Molecular
Origin of the Voltage Dependence of G-Protein Regulation of P/Q-type Ca²⁺ Channels

Yun Zhang,¹ Yu-hang Chen,¹ Saroja D. Bangaru,¹ Linling He,¹ Kathryn Abele,¹ Shihori Tanabe,² Tohru Kozasa,² and Jian Yang¹

¹Department of Biological Sciences, Columbia University, New York, New York 10027, and ²Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612

Correspondence should be addressed to Jian Yang, Department of Biological Sciences, 917 Fairchild Center, MC2462, Columbia University, New York, NY 10027. Email: jy160{at}columbia.edu

G-protein (Gβ)-mediated voltage-dependent inhibition of N- and P/Q-type Ca²⁺ channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gβ from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca²⁺ channel β subunit (Ca_vβ) and a rigid -helical structure between the -interacting domain (AID), the primary Ca_vβ docking site on the channel ₁ subunit, and the pore-lining IS6 segment. Gβ inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca_vβ were produced by washing out a mutant Ca_vβ with a reduced affinity for the AID. These β-less channels were still inhibited by Gβ, but without any voltage dependence, indicating that Ca_vβ is indispensable for voltage-dependent Gβ inhibition. A truncated Ca_vβ containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gβ inhibition. Gβ did not alter inactivation properties, and channels recovered from Gβ inhibition exhibited the same activation property as un-inhibited channels, indicating that Gβ does not dislodge Ca_vβ from the inhibited channel. Furthermore, voltage-dependent Gβ inhibition was abolished when the rigid -helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca_vβ regulation of channel gating, revealing a pivotal role of this rigid -helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gβ-binding pocket through a rigid -helix induced partly by the Ca_vβ GK domain, causes the dissociation of Gβ and is fundamental to voltage-dependent Gβ inhibition.

Key words: Gβ; voltage-dependent modulation; β subunit; helix; inhibition; patch clamp

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Received March 28, 2008; revised Nov. 12, 2008; accepted Nov. 13, 2008.

Correspondence should be addressed to Jian Yang, Department of Biological Sciences, 917 Fairchild Center, MC2462, Columbia University, New York, NY 10027. Email: jy160{at}columbia.edu

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