To assess the biological consequences of decreased numbers of BS in a more quantitative manner, we determined the real-time electrophysiological effects of the holotoxin variants on polarized human intestinal cells (T84 cell line) by measuring the short circuit current required to eliminate the potential difference induced by the cAMP-dependent Clsecretory response resulting from CT-A1-mediated ADP ribosylation of Gs␣ and constitutive activation of adenylate cyclase. Figure 4A to 4F shows the results of these experiments. Panel A shows that loss of even a single GM 1 BS attenuated the activity of cholera toxin to some degree, an effect which increased as the number of native GM 1 BS was lost. The time of onset of intoxication was also delayed as the number of native GM 1 BS decreased. Nevertheless, toxin with a single native GM 1 BS had clearly detectable activity. As expected, the G33D holotoxin with no native GM 1 BS had almost no activity over the time frame of the experiment: 1.5% of wt toxin signal over baseline at 90 min and less than 9% of wt signal at 120 min. Since these variants have differing numbers of C-terminal GSH6 tags (zero, one, or two), we also examined the effect that the number of tags had on the activity of wt holotoxin with five BS (Fig. 4B) . The presence of the tags modestly affected both the time of onset of intoxication, a measure of toxin-GM 1 trafficking from the PM to the ER of host cells, and the rate of increase of I sc . The effect was greater for doubly tagged holotoxin, although it eventually showed a similar maximal I sc . To control for these effects of the B-subunit tags, toxins with different numbers of native GM 1 BS but the same number of tags were compared. For these analyses, data were normalized by setting the maximal signal for the five native GM 1 BS toxins in each comparison to 1.00 (Fig. 4C to F) . For the toxin variants with one tag on the B subunit, the holotoxin with four native GM 1 BS exhibited a slight (25%) attenuation of toxicity relative to holotoxin with five native GM 1 BS, seen as a decrease in maximal I sc but with no delay in onset of intoxication (Fig. 4C) . A similar result (30% decrease of maximal I sc ) was seen for comparisons of the holotoxin variants with three or five native GM 1 BS and two CTB-GSH6-tagged subunits (Fig. 4D) . When the number of native GM 1 BS was reduced to two or one in doubly or singly wt B-subunit-tagged holotoxin variants, the peak I sc decreased by more than 50% and also the onset of intoxication was delayed ( Fig. 4E and 4F) , suggesting defects in entry of CT into the cell or transport to the ER (or both).