Figure 6: Regulation of TRPML1 by SMs and sphingomyelinases.

(a) SF-51-activated whole-cell I_ML1−4A was inhibited by SMs (20 μM) at low extracellular pH in ML1-4A-expressing HEK293T cells. (b) Insensitivity of whole-cell I_ML1−Va to SMs (60 μM). (c) Lack of effect of ceramide (Cer, 20 μM) on whole-cell I_ML1−4A. (d) Summary of the effects of various sphingolipids on whole-cell I_ML1−4A in ML1-4A-expressing HEK293T cells. PC: phosphocholine. (e) SF-51-activated whole-cell I_ML1−4A was potentiated by sphingosine (Sph; 5 μM) at low external pH in ML1-4A-expressing HEK293T cells. (f) The effects of SMase (50 ng ml⁻¹) treatment on I_ML1−4A at low external pH. I_ML1−4A was elicited by repeated voltage ramps (−140 to + 140 mV; 400 ms) with a 4-s interval between ramps. Current amplitudes at −140 mV were used to plot the time dependence. (g) Representative traces of I_ML1−4A before (black) and after (dark yellow) SMase treatment at two different time points, as shown in f. (h,i) SMase (50 ng ml⁻¹) treatment alone had no (six out of seven cells; see h for an example) or small (one out of seven cells; i) activation effect on basal whole-cell I_ML1−4A in ML1-4A-expressing HEK293T cells. (j) The effects of SMase (50 ng ml⁻¹) treatment on SF-51-activated I_ML1−4A at neutral external pH (pH 7.4). (k) The potentiation effect of SMase was more dramatic at pH 4.6, but abolished by heat inactivation of the enzyme activity. HI-SMase: heat-inactivated SMase. For panels d and k, data are presented as the mean±s.e.m.; the n numbers are in parentheses. Statistical comparisons were made using analysis of variance: *P<0.05.