C1C-3 CHLORIDE ION CHANNELS IN VASCULAR SMOOTH MUSCLE

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Chloride (Cl) currents contribute to both stretch and agonist- induced depolarization of vascular smooth muscle (VSM) cells. Changing the Cl gradient, interfering with C1 transport, or blocking Cl channels all alter VSM contractile responses to norepinephrine. The Cl channels responsible for this effect are regulated by nitric oxide. This may represent a new mechanism for the control of VSM contractility. Myogenic tone is also inhibited by C1 channel blockers and sensitive to changes in the C1 gradient. We have shown by Northern blotting that in cultured human VSM cells (aortic and coronary), by far the most highly expressed C1 channel gene is C1C-3. Vascular expression of C1C-3 was confirmed by in situ hybridization in human lung. It has recently been shown that expression of a C1C-3 clone in NIH/3T3 cells produces a volume-activated chloride current (IC1vol) which is inhibited by activators of protein kinase C. It is not known if a response to swelling translates to sensitivity to mechanical stretch or if these channels can be activated by contractile agents. We have found that the C1C-3 gene is alternatively spliced in mice and rats so that exon 2 is excluded (CIC-3Short) while human mRNA always includes this sequence (C1C-3Long). This is important because exon 2 contains an in frame ATG producing a C1C-3 protein which is 58 amino acids longer at the amino terminus than the previously expressed clone which produced IC1vol. The function of these 58 amino acids needs to be determined. We propose to; 1) use perforated patch-clamp recording to define the contribution of the calcium-activated and swelling-activated chloride currents to catecholamine-mediated depolarization of mouse aortic VSM cells. We will then determine if, and how, these currents are regulated by nitric oxide, 2) study the volume-activated chloride currents produced by three different cell types which either completely lack C1C-3 (mouse embryonic stem cells with C1C-3 knocked out) or have been made to overexpress C1C-3L or C1C-3S (NIH/3T3 cells, Fisher Rat Thyroid epithelial cells), and 3) assess the function of C1C-3 in intact murine blood vessels by using transgenic technology to overexpress C1C-3 behind a VSM-specific promoter (SM22alpha). These studies will define how VSM Cl channels are activated and regulated and how C1C-3 contributes to blood vessel function and determination of blood pressure. They will also further our understanding of the basic biophysical characteristics of C1C-3. An understanding of these issues may allow us to design new pharmacological approaches to the control of vascular function.