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Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities
1Department of Biology, State University of New York, Albany, New York 12222, USA. wang@zeus.bwh.harvard.edu
Abstract:
Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.
Insights
Batrachotoxin (BTX) and local anesthetics (LAs) interact with voltage-gated sodium channels. Researchers identified key residues in the D3-S6 segment crucial for both BTX and LA binding, impacting channel function.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Voltage-gated sodium channels (Nav channels) are crucial for nerve impulse propagation.
- Batrachotoxin (BTX) locks Nav channels in an open state, while local anesthetics (LAs) block sodium ion conductance.
- Previous studies mapped BTX and LA binding sites to common residues in D1-S6 and D4-S6 segments of the Nav channel alpha-subunit.
Purpose of the Study:
- To investigate if additional residues in the D3-S6 segment of the rat muscle mu1 Nav channel are involved in BTX and LA binding.
- To identify specific amino acid residues critical for the interaction of BTX and LAs with Nav channels.
Main Methods:
- Site-directed mutagenesis was used to substitute individual residues with lysine in the D3-S6 segment (F1274 to N1281) of the rat muscle mu1 Nav channel.
- Mutant channels were expressed in mammalian cells.
- The effects of BTX and bupivacaine on mutant channel function, including activation, inactivation, and binding affinity, were assessed using electrophysiological techniques and radioligand binding assays.
Main Results:
- Two mutant channels, mu1-S1276K and mu1-L1280K, exhibited complete resistance to 5 microM BTX during repetitive pulses.
- These mutants displayed significantly altered activation and/or fast inactivation gating compared to wild-type channels.
- Mutants showed a 10-20 fold reduction in bupivacaine affinity for the inactivated state, with only a twofold change for the resting state.
Conclusions:
- Residues mu1-S1276 and mu1-L1280 in the D3-S6 segment are critical for both BTX and LA binding interactions.
- Local anesthetics likely interact with these D3-S6 residues, along with residues in D1-S6 and D4-S6, when the Nav channel is in its inactivated state.
- The findings support a state-dependent binding model for LAs, influencing S6 segment alignment within the Nav channel.