Custom Distinctions in the Interaction of G-protein β Subunits with N-type (CaV2.2) Versus P/Q-type (CaV2.1) Calcium Channels
| Autor: | David T. Yue, Jenafer Evans, Henry M. Colecraft, Heather L. Agler |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Physiology
G protein GTP-Binding Protein beta Subunits Kidney Transfection medicine.disease_cause Article Cav2.1 Calcium Channels N-Type medicine Animals Humans Point Mutation voltage-dependent regulation α1A and α1B Mutation Alanine biology Voltage-dependent calcium channel Point mutation mathematical modeling Models Theoretical Receptor–ligand kinetics Electrophysiology channel modulation Biochemistry biology.protein Biophysics G proteins |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| DOI: | 10.1085/jgp.200208770 |
| Popis: | Inhibition of N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gbetagamma complex (Gbetagamma). Far less is known about the important interaction sites on Gbetagamma itself. Here, we developed a novel electrophysiological paradigm, "compound-state willing-reluctant analysis," to describe Gbetagamma interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gbetagamma with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gbeta1, at residues known to interact with Galpha. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gbetagamma for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for "steering" or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel. |
| Databáze: | OpenAIRE |
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