| Summary: | 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.
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