Development of resistance to paralytic shellfish toxins in the softshell clam, Mya arenaria, via a single mutation in the sodium channel pore region: III. Electrophysiological studies

Vera L. Trainer¹, Keiichi Konoki², William A. Catterall², Todd Scheuer², Laurie Connell³, Monica Bricelj⁴

¹NOAA Fisheries, Marine Biotoxin Program, Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle, WA, 98112, USA (Vera.L.Trainer@noaa.gov)
²University of Washington, Department of Pharmacology, Box 357280, Seattle, WA, 98195, USA (konoki@u.washington.edu, wcatt@u.washington.edu, scheuer@u.washington.edu)
³School of Marine Sciences, University of Maine, Orono, Maine, 04469, USA (Laurie_Connell@umit.maine.edu)
⁴National Research Council Canada, Halifax, Nova Scotia, B3H 3Z1, Canada (monica.bricelj@nrc.ca)

Populations of the softshell clam, Mya arenaria, that have different long-term histories of exposure to paralytic shellfish toxins (PSTs) in the field are distinguished by their relative sensitivity or resistance to these toxins (Bricelj et al. abstract). Here we demonstrate that a single mutation at the saxitoxin binding site in the pore region of the voltage-sensitive sodium channel (Connell et al. abstract) confers resistance to Mya arenaria. The plasmid pCDM8 Nav1.2, which encodes the entire rat brain sodium channel, was used to create mutant sodium channel cDNA. A human embryonic kidney cell line (tsA-201) was transfected with the plasmid DNA followed by whole cell voltage clamp recording on cells expressing the channels. The peak sodium current conducted by wild type and mutant sodium channels measured during a single pulse to -10 mV was inhibited by tetrodotoxin (TTX) and saxitoxin (STX) in a dose dependent manner. The IC50 of those toxins applied to wild type channels was ca. 10 nM and 1 nM respectively, equivalent to published values. Conversely, the sodium current in the mutant channel was 3000-fold more resistant to block by TTX and STX with IC50 values of ca. 30 µM and 3 µM, respectively, demonstrating the importance of a single amino acid residue in determining the affinity of these toxins to their receptor site in the sodium channel pore. This study describes the molecular basis of adaptation to PSTs by M. arenaria via a single mutation in the sodium channel pore region allowing resistant individuals to survive toxin exposure and retain significantly higher levels of PSTs, thereby resulting in an increased threat to public health.