Cardiovascular disease is an increasing problem in the UK, resulting in over 159,000 deaths each year. Cardiovascular disease has been linked to disturbed osmosensing, known to effect sympathetic activity altering blood pressure (BP) and heart rate (HR)[1]. The hypothalamus is responsible for maintaining plasma osmolality within a narrow range (∼290-300mOsm)[2]. However, the mechanisms involved have yet to be established. The paraventricular nucleus (PVN) of the hypothalamus is thought to have a role in osmoregulation; hypertonic solutions increase electrical activity of PVN neurones[3]. Recent studies implicate the transient receptor potential vanilloid channel (TRPV4) and calcium activated potassium channels (KCa) as possible candidates for osmosensing [4]. Using a combination of in vivo, in vitro and in silico we have investigated a possible functional coupling of TRPV4 and KCa in PVN cardiovascular control neurone regulation in response to osmotic challenge. Mice were anaesthetized intraperitoneally with urethane-chloralose (1.4-2.2mg/kg-7-11µg/kg). BP was measured by arterial cannulae and compounds applied by intracerebroventricular (ICV) injection. Cellular mechanisms were investigated using the NEURON simulation environment and experimentally by cell-attached patch-clamp recording in mouse PVN hypothalamic brain slices. Results are given as mean±SEM; in vivo and in vitro significances were assessed by two-way ANOVA with post-hoc Tukey comparison and Student’s paired t-tests respectively. ICV injection of hypotonic saline led to a rapid decrease in BP, whereas isotonic saline had no effect (-9±2mmHg vs. -2±1mmHg; n=6; p<0.01). These decreases in BP were abolished by the selective TRPV4 inhibitor RN1734 (-1±1mmHg; n=6). Using action currents to indicate action potential frequency in PVN brain slice experiments, we established that the osmotic effect on action potential frequency is dependent upon both TRPV4 channels (hypotonic: 70±14% reduction vs. hypotonic with RN1734: 45±15%; n=6; p<0.05) and small conductance KCa (SK) (hypotonic: 79±10% reduction vs. hypotonic with the SK channel inhibitor UCL-1684: 37±7%; n=5; p<0.01), but no significant effects of the intermediate and large conductance KCa channel inhibitors TRAM-34 and iberiotoxin. In order to verify the presence of TRPV4 within the parvocellular PVN, single channel activity was recorded. A population of ion channels was identified with a mean slope unitary conductance of 57±7pS and reversal potential of -5±3mV (n=6); indicative of a non-selective cation channel. This channel activity was seen in 50% of patches (8/16), with a mean open probability (Po) of 0.1±0.0 at -40mV. Po increased by 48±9% (n=4) upon addition of the TRPV4 agonist 4αPDD. These results strongly suggest that central osmolality changes modulate the cardiovascular system by functional coupling of TRPV4 and small conductance KCa channels in the parvocellular PVN.