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Ca²⁺ Signals Laboratory, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Address correspondence to David T. Yue, Ca²⁺ Signals Laboratory, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Ave. Baltimore, MD 21205. Fax: (410) 614-8269; E-mail: dyue{at}bme.jhu.edu

Inhibition of N- (Ca_v2.2) and P/Q-type (Ca_v2.1) 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 Gß complex (Gß). Far less is known about the important interaction sites on Gß itself. Here, we developed a novel electrophysiological paradigm, "compound-state willing-reluctant analysis," to describe Gß 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 Gß 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 Gß₁, at residues known to interact with G. 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 Gß 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.

Key Words: _1A and _1B • channel modulation • voltage-dependent regulation • mathematical modeling • G proteins

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