In an age where multidrug resistance is becoming more prevalent, there is need for novel drug targets. Gram-negative bacteria contain porins in the outer membrane, and control influx of antibiotics. The alteration in the expression of porins is one of the methods contributing to multidrug resistance. In Escherichia coli, the well understood two-component system (TCS) EnvZ/OmpR regulates outer membrane porins OmpC and OmpF. EnvZ exhibits cytoplasmic osmosensing and periplasmic allosteric interaction with MzrA to change the ratio of EnvZ kinase and phosphatase activity. To monitor the position and dynamics of helices in signal transduction, we performed in vivo disulphide crosslinking within the transmembrane domain (TMD) between EnvZ and MzrA across ~250 Cys substituted pairings. We found Cys crosslinking between EnvZ and MzrA at the cytoplasmic and periplasmic ends of the TMD, indicating these residues face towards one other in a close enough proximity for crosslinking to form.  We also analysed in vivo signal output across 15 Cys substitutions in the cytoplasmic domain of EnvZ and the increase in signal output in the N-terminal towards the TM region suggests the presence of a water filled hemichannel. Furthermore, we propose that these water filled hemichannels are a conserved motif in sensor kinases and promote transmembrane communication.