Effects of intracellular magnesium on large conductance, calcium-activated potassium channels in rat cerebrovascular smooth muscle cells
Creators: name: Zhang, Xian
Publisher: name: University of British Columbia
Year: 2009

description: Increasing the plasma concentration of Mg²⁺ above normal levels results in the dilation of cerebral arteries. Conversely, an abnormally low level of Mg²⁺ in plasma has been shown to increase cerebrovascular tone and to induce calcium-mediated vasospastic responses in cerebral vessels. It is known that large conductance, calcium-activated potassium channels (BK channels) play an important role in the regulation of myogenic tone in cerebrovascular smooth muscle cells (CVSMCs). In the vascular smooth muscle cells of systemic vessels, the properties of BK channels are themselves modulated by the intracellular concentration of free magnesium ions, [Mg²⁺]i. In CVSMCs, [Mg²⁺]i increases on elevation of the plasma concentration of magnesium ions. It therefore seemed possible that the vasodilatory effect of high plasma Mg²⁺ levels could result in part from direct, intracellular actions of the cation on BK channel function. The present project was undertaken to test this hypothesis. CVSMCs from the basilar, middle and posterior cerebral arteries of adult Wistar rats were dispersed using collagenase and trypsin and maintained in vitro for 48 hours prior to use. Recordings of single BK channel currents were made at room temperature (20 - 24 °C) from inside-out membrane patches excised from these cells, using a List EPC-7 patch clamp amplifier. Concentrations of Mg²⁺ f higher than 1 mM reversibly reduced the amplitude of currents flowing through open BK channels. In this action, Mg² f behaved as a fast blocker, reducing BK channel currents in a concentration and a voltage-dependent manner. The blocking effect of Mg²⁺j was well described by the Woodhull model, which postulates the physical occlusion of the channel pore by a penetrating ion. However, the affinity and voltage-dependence of Mg²⁺i block were found to be dependent on the concentration of free intracellular calcium ions, [Ca²⁺]i, bathing the cytoplasmic face of membrane patches. Ca²⁺i may stabilize a conformation of the BK channel protein in which the Mg²⁺i binding sites are relocated closer to the inner membrane surface, reducing the voltage-dependency of Mg²⁺i block. In the presence of 1 ɥM [Ca²⁺]i, Mg²⁺i enhanced the open probability (P₀) of BK channels in a concentration-dependent manner, this effect being evident at the physiologically relevant concentration of 0.5 mM [Mg²⁺]i. Mg²⁺i shifted the Boltzmann curve relating P₀ to membrane potential leftwards on the voltage axis, without any change in its slope. The enhancing effect of Mg²⁺ on P₀ was, therefore, not itself a voltage-dependent process. These results suggest that the sites which Mg²⁺i must occupy to increase P₀ are distinct from those which are involved in blocking current flow through the open channel. Quantitative considerations suggest that the blocking action of Mg²⁺i on BK channel currents is unlikely to play a significant role in modulating channel function under physiological conditions. However, physiological levels of Mg²⁺i would tonically facilitate the effect of Ca²⁺i on BK channel activation. The abnormally high or low levels of Mg²⁺ associated with hyper- or hypomagnesemia may also contribute to the dilation or contraction of cerebral vessels seen under these two conditions.