A subset share characteristics that make them effective as antiarrhythmic drugs, ie, they exhibit high affinity, use-dependent block of Na current (I Na ) at high heart rates. Despite extensive study, there remains uncertainty regarding how observed block relates to specific drug-channel conformations. Several vocabularies have emerged to describe block, which in general, have their basis in kinetic models of Na channel gating and assume preferential binding to one or more states that produce no 1 or altered 2 gating. Recent availability of crystal structures in combination with mutagenesis data now allow for linking electrophysiological data, kinetic states, and drug block to specific channel conformations.It is generally accepted that lidocaine and lidocaine-like drugs bind in the inner pore of voltage-gated Na channels. Scanning mutagenesis studies with various Na channel isoforms and multiple lidocaine-like drugs have identified only one amino acid residue, a phenylalanine (Phe) in domain IV, S6 (DIVS6), which, when mutated, alters use-dependent drug affinity by more than ten-fold. When this Phe (1759 in Na V 1.5) is mutated to nonaromatic residues 3-8 or to unnatural amino acids with different electron withdrawing capabilities 9 the mutated channel shows a marked decrease in high-affinity LA block. Homology modeling with K channels predicts that this Phe faces the pore just below the selectivity filter. 10,11 This orientation of Phe is supported by the finding that its cysteine mutant is accessible to MTS reagents applied from inside the pore when the channel is maintained in an open state. 12 Furthermore, it has been shown by us 13 and others 14 that use-dependent block is intimately associated with altered movements of the structurally distant S4 segments in domains III and IV.Block assayed from negative holding potentials at low rates of stimulation is affected very little by channel mutations in contract to their effects on use-dependent block. This lower affinity block is usually called tonic block, although it has also been called rested-state block (or closed-state block)

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Cerebral vasospasm is a transient, delayed constriction of cerebral arteries that occurs after subarachnoid hemorrhage (SAH). Smooth muscle cells show impaired relaxation after SAH, which may be caused by a defect in the ionic mechanisms regulating smooth muscle membrane potential and Ca(2+) permeability. We tested this hypothesis by examining changes in expression of mRNA and protein for ion channels in the basilar arteries of dogs after SAH using quantitative real-time polymerase chain reaction (PCR) and western blotting. SAH was associated with a significant reduction in basilar artery diameter to 41 +/- 8% of pre-SAH diameter (P < 0.001) after 7 days. There was significant downregulation of the voltage-gated K(+) channel K(v) 2.2 (65% reduction in mRNA, P < 0.001; 49% reduction in protein, P < 0.05) and the beta1 subunit of the large-conductance, Ca(2+) - activated K(+) (BK) channel (53% reduction in mRNA, P < 0.02). There was no change in BK beta1 subunit protein. Changes in mRNA levels of K(v) 2.2 and the BK-beta1 subunit correlated with the degree of vasospasm (r(2) = 0.490 and 0.529 respectively, P < 0.05). The inwardly rectifying K(+) (K(ir)) channel K(ir) 2.1 was upregulated (234% increase in mRNA, P < 0.001; 350% increase in protein, P < 0.001). There was no significant change in mRNA expression of L- type Ca(2+) channels and the BK-alpha subunit. These data suggest that K(+) channel dysfunction may contribute to the pathogenesis of cerebral vasospasm.

Cerebral vasospasm is a major cause of morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). It is a sustained constriction of the cerebral arteries that can be reduced by endothelin (ET) receptor antagonists. Voltage-gated Ca(2+) channel antagonists such as nimodipine are relatively less effective. Endothelin-1 is not increased enough after SAH to directly cause the constriction, so we sought alternate mechanisms by which ET-1 might mediate vasospasm. Vasospasm was created in dogs, and the smooth muscle cells were studied molecularly, electrophysiologically, and by isometric tension. During vasospasm, ET-1, 10 nmol/L, induced a nonselective cation current carried by Ca(2+) in 64% of cells compared with in only 7% of control cells. Nimodipine and 2-aminoethoxydiphenylborate (a specific antagonist of store-operated channels) had no effect, whereas SKF96365 (a nonspecific antagonist of nonselective cation channels) decreased this current in vasospastic smooth muscle cells. Transient receptor potential (TRP) proteins may mediate entry of Ca(2+) through nonselective cationic pathways. We tested their role by incubating smooth muscle cells with anti-TRPC1 or TRPC4, both of which blocked ET-1-induced currents in SAH cells. Anti-TRPC5 had no effect. Anti-TRPC1 also inhibited ET-1 contraction of SAH arteries in vitro. Quantitative polymerase chain reaction and Western blotting of seven TRPC isoforms found increased expression of TRPC4 and a novel splice variant of TRPC1 and increased protein expression of TRPC4 and TRPC1. Taken together, the results support a novel mechanism whereby ET-1 significantly increases Ca(2+) influx mediated by TRPC1 and TRPC4 or their heteromers in smooth muscle cells, which promotes development of vasospasm after SAH.

Delayed cerebral vasospasm after subarachnoid hemorrhage is primarily due to sustained contraction of arterial smooth muscle cells. Its pathogenesis remains unclear. The degree of arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance (GK). Here, we identified the main voltage-gated K+ (Kv) channels contributing to outward delayed rectifier currents in dog basilar artery smooth muscle as Kv2 class through a combination of electrophysiological and pharmacological methods. Kv2 current density was nearly halved in vasospastic myocytes after subarachnoid hemorrhage (SAH) in dogs, and Kv2.1 and Kv2.2 were downregulated in vasospastic myocytes when examined by quantitative mRNA, Western blotting, and immunohistochemistry. Vasospastic myocytes were depolarized and had a smaller contribution of GK toward maintenance of their membrane potential. Pharmacological block of Kv current in control myocytes mimicked the depolarization observed in vasospastic arteries. The degree of membrane depolarization was found to be compatible with the amount of vasoconstriction observed after SAH. We conclude that Kv2 dysfunction after SAH contributes to the pathogenesis of delayed cerebral vasospasm. This may confer a novel target for treatment of delayed cerebral vasospasm.