Fig. 5.

Effect of short hairpin (sh) RNA knockdown of TRPM4 on Ca²⁺-activated current in apical membrane patches from mIMCD-3 cells. A: RT-quantitative (q) PCR for Trpm4 mRNA expression. RT-qPCR was performed to determine Trpm4 mRNA expression relative to the reference genes B2m and Sdha. Stable transduction of mIMCD-3 cells with Trpm4 shRNA (knockdown; KD) reduced Trpm4 mRNA expression compared with wild-type (WT) cells or cells treated with a non-target control (NTC) shRNA. The percent reductions compared with WT were 59 and 48% using the B2m and Sdha reference genes, respectively (n = 3 passages). *Significantly different from WT and NTC for a given reference gene (P < 0.007, Tukey test). B: the current-voltage relationship was determined in cytoplasmic solution with 1 mM Ca²⁺ (Table 1) in apical membrane patches from cells in which TRPM4 was knocked down (KD) and in NTC. Pipettes contained the standard external solution. Leak current was subtracted as described in materials and methods. C: mean Ca²⁺-activated current at +100 mV in patches from each of the two cell types. Current-voltage relationships and currents are averages from 12 (KD) and 14 (NTC) patches.