Block of Nachbac by Cadmium and Lanthanum Ions

Abstract

The prokaryotic, voltage-gated sodium channel, NaChBac, has features common to both eukaryotic Nav and Cav channels, but also differs in important details. NaChBac assembles as a homo-tetramer, and it achieves Na+-selective conduction with a pseudo-symmetric selectivity filter possessing a 4-glutamate motif (EEEE), which is common also to eukaryotic Cav channels, but is distinctly different from the asymmetric selectivity filter (DEKA) of eukaryotic Nav channels. Block by impermeant, or weakly permeant, ions offers functional clues as to the nature and location of ion-binding sites within the channel's conducting pathway. Here, we examine block by a transition metal (Cd2+), or a lanthanide (La3+), in the extracellular solution. Whole-cell patch-clamp experiments were performed using tsA201 cells transiently expressing NaChBac. Onset of block and washout were observed using repeated depolarizations to −10 mV, from a holding potential of −120mV (IC50s (mM, mean±SEM): Cd, 2.0±0.06; La, 0.81±0.03. Two analyses examined voltage dependence of block and provided estimates of the “apparent electrical distance”, δ, from external solution to the blocking site based on the analysis of Woodhull (J. Gen. Physiol., 1973). First, peak current-voltage relations were determined in the presence and absence of the blocker. Second, instantaneous I-V relations allowed calculation of fractional block of the conductance activated by a fixed, activating prepulse. In each analysis, a fit of fractional block, fb, vs V yielded an estimate of δ, and the IC50(V=0). IC50 values were similar to those obtained directly from dose-response data (see above). Values for δ appeared to vary slightly with the method, but indicated only weak V dependence of block: Cd, 0.02 - 0.08; La, 0.02 - 0.13. These data are consistent with a superficial binding site, and only weak coupling of block to movements of permeant ions within the channel. Supported by: MOP-10053 (CIHR).