Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Abstract

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca 2+ ] cyt ), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/ vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone. Video LinkThe video component of this article can be found at http://www.jove.com/video/4263/ Protocol Surgical Excision of the Small Intestinal Mesenteric Vascular Arcade1. Anesthetize a 300-400 g Sprague-Dawley rat with isoflurane (4%) administered by inhalation. 2. Perform a midline laparotomy to expose the small intestinal mesentery. Exteriorize the small and large intestine through the abdominal incision with great care to avoid trauma to the exposed bowel and mesentery. Gently fan the mesentery out over sterile gauze. 3. Surgically excise the small intestine and a portion of the large intestine, including the caecum (blood vessels carefully cut along the margins and at the origins from the primary branches of the superior mesenteric artery/vein). 4. Transfer the excised tissue to a beaker containing ice cold dissecting solution ( Table 1). 5. Following excision of the intestine, the rat should be humanely euthanized while under anesthesia. Dissection and Cleaning of Arteries1. Transfer the excised intestine and mesenteric arcade to a cooled dissecting chamber (4-5 °C) containing dissecting solution. The chamber consists of a 100-mm glass Petri dish with a SYLGARD 184 elastomer base to accommodate dissecting pins; ...

Involvement of transglutaminase 2 and voltage‐gated potassium channels in cystamine vasodilatation in rat mesenteric small arteries

“…XE991 can also attenuate K v 1.2/K v 1.5 and K v 2.1/K v 9.3 channels (Zhong et al, 2010), and high extracellular potassium had a more pronounced inhibitory effect on cystamine relaxation than XE991 and linopirdine. Therefore, other approaches, for example, perforated patch configuration, will be required to further delineate whether other K v channel subtypes are involved (Brueggemann et al, 2012), but our present findings that both XE991 and linopirdine inhibit cystamine-induced relaxation and increase in current suggest that K v 7 channels contribute to cystamineinduced relaxation in rat mesenteric arteries.…”

KV7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric KV7.4/KV7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function

“…To effectively isolate K V 7 currents, recordings were made in the presence of the selective BK channel inhibitor paxilline (1 mM) and GdCl 3 (50 mM), an inhibitor of nonselective cation and L-type voltage-gated Ca 21 channels. Further, the more depolarized holding potential of 210 mV that we used ensures inactivation of other non-K V 7 K 1 channels, such as inward-rectifying K 1 channels (Brueggemann et al, 2012b). ML213 (10 mM) caused a significant increase in the amplitude of whole-cell K V 7 currents at all voltages from 250 to 140 mV (n 5 7, N 5 5; P , 0.05) (Fig.…”

“…8 and 9). The use of a more depolarized holding potential (210 mV) in our study ensured inactivation of other non-K V 7 K 1 channels, such as inward-rectifying K 1 channels (Brueggemann et al, 2012b). Also, at a 210-mV holding potential, some of the K V 7 channels are open and generate quantifiable currents, which are not contaminated by the presence of nonselective cation currents due to the inclusion of GdCl 3 .…”

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

“…The perforated patch-clamp technique has been a mainstay electrophysiological approach for over a quarter of a century. Several publications provide details of the technical considerations 69 , 70 , 71 , 72 , 73 . Cell perforation can be obtained with amphotericin-B, nystatin, gramicidin, or β-escin (see reference 32 for overview of each).…”