Defining mechanisms of CaV1.2 dysregulation in hypertension

About This Grant

Hypertension is a major risk factor for heart disease, kidney disease and stroke, causing death and disability in alarming numbers of Americans. Vascular resistance, largely determined by vasomotor tone in splanchnic resistance vessels, is a major determinant of blood pressure. CaV1.2, the voltage activated calcium channel is essential for maintaining blood pressure; when deleted from vascular smooth muscle in mice, marked hypotension results. Rats genetically prone to hypertension have excessive vasoconstriction and exaggerated responses to vasoactive hormones, associated with abnormally active CaV1.2. Many studies have shown CaV1.2 currents are increased in the vascular smooth muscle of Spontaneously hypertensive (SHR) rats. Some studies attribute this to an increase in channel numbers; however, others have shown in single-channel recordings that CaV1.2 has increased opening probability. SHR rats have atypical alternative splicing in the transcripts that encode CaV1.2, though it is unclear if this is a cause or a consequence of hypertension in these animals. Inclusion of Exon 9* is of particular interest since channels with this exon are upregulated in the heart in other cardiovascular diseases and these cardiac channels with Exon 9* also have high open probability. In Aim 1 of this study, through crossing a transgenic mouse with doxycycline inducible expression of CaV1.2 containing either typical Exon 9 or Exon 9*, with mice that express rtTA in smooth muscle, one can examine the effects of increasing the proportion of channels containing Exon 9*. By measuring blood pressure and contractility in vascular smooth muscle, it will be possible to tell if increasing expression of channels with Exon 9* is sufficient to generate hypertension. Aim 2 will test the hypothesis that increased vascular tone in SHR rats results from increased recruitment of channel activators or reduced targeting of channel inhibitors to the channel complex. Examples of activators include effectors of vasoconstricting hormones that signal through GPCRs and ultimately PKC, while examples of channel inhibitors include effectors of endothelial relaxing factors like the Cyclic GMP dependent protein kinase or PKG. Because the cell lysis necessary to extract protein from blood vessels disrupts large channel complexes, especially those in lipid membranes, where CaV1.2 is located, standard proteomics techniques have limited resolution to answer such questions. But by targeting a biotin ligase to the channel complex, it is possible to covalently tag interacting proteins. CaV1.2 b2 subunits have nanomolar affinity for the CaV1.2 pore. By injecting SHR rats and healthy rats with AAV5 containing CaV1.2 b2 subunit fused to TurboID, under the control of a smooth muscle promoter, one can biotin label proteins in the vicinity of the channel in blood vessels of live rats. After purifying biotinylated proteins, with TMT mass spectrometry we will quantify channel interactors based on their proximity to the channel, including known activators and inhibitors of CaV1.2. The proposed experiments will determine if atypical splicing or dysregulation of CaV1.2 interactors contribute to the severely increased blood pressure in SHR rats.