1 To assess the action of nitric oxide (NO) and NO-donors on K + current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2 Inside-out patches contained channels, the open probability of which increased with [Ca 2+ ] i . The channels had a linear slope conductance of 212+5 pS (n=12) in symmetrical (140 mM) K + solutions which reversed in direction at 4.4 mV. In addition, the channels showed K + selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K + . Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK Ca . 3 In ®ve out of six inside-out patches, NO (45 or 67 mM) produced an increase in BK Ca activity. In inside-out patches, BK Ca activity was also enhanced in some patches with 100 or 200 mM 3-morpholinosydnonimine (SIN-1) (4/11) and 100 mM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may re¯ect variability in the release of NO from these compounds. 4 In inside-out patches, 100 mM SIN-1 failed to increase BK Ca activity (in all 4 patches tested), while at a higher (500 mM) concentration SIN-1 had a direct blocking e ect on the channels (n=3). NO applied directly to inside-out patches increased (P50.05) BK Ca activity in two patches. 5 In the majority of cells (6 out of 7), application of NO (45 or 67 mM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not a ected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in wholecell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 mM). With SIN-1, the increase in current was blocked with the BK Ca channel blocker, iberiotoxin (50 nM). 6 With conventional whole-cell voltage clamp, the increase in the outward K + current evoked with SIN-1 (50 ± 300 mM) showed considerable variability. Either no e ect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+10.2% was recorded at 40 mV. 7 In cell-attached patches, large conductance voltage-dependent K + channels were stimulated by SIN-1 (100 mM) applied to the cell (n=5 patches). 8 These data indicate that NO and its donors can directly stimulate BK Ca activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK Ca channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.

Smooth muscle cells isolated from the secondary and tertiary branches of the rabbit mesenteric artery contain large Ca2+-dependent channels. In excised patches with symmetrical (140 mm) K+ solutions, these channels had an average slope conductance of 235 +/- 3 pS, and reversed in direction at -6.1 +/- 0.4 mV. The channel showed K+ selectivity and its open probability (Po) was voltage-dependent. Iberiotoxin (50 nm) reversibly decreased Po, whereas tetraethylammonium (TEA, at 1 mm) reduced the unitary current amplitude. Apamin (200 nm) had no effect. The channel displayed sublevels around 1/3 and 1/2 of the mainstate level. The effect of [Ca2+] on Po was studied and data fitted to Boltzmann relationships. In 0.1, 0.3, 1.0 and 10 microM Ca2+, V1/2 was 77.1 +/- 5.3 (n = 18), 71.2 +/- 4.8 (n = 16), 47.3 +/- 10.1 (n = 11) and -14.9 +/- 10.1 mV (n = 6), respectively. Values of k obtained in 1 and 10 microM [Ca2+] were significantly larger than that observed in 0.1 microM [Ca2+]. With 30 microM NS 1619 (a BKCa channel activator), V1/2 values were shifted by 39 mV to the left (hyperpolarizing direction) and k values were not affected. TEA applied intracellularly, reduced the unitary current amplitude with a Kd of 59 mm. In summary, BKCa channels show a particularly weak sensitivity to intracellular TEA and they also display large variation in V1/2 and k. These findings suggest the possibility that different types (isoforms) of BKCa channels may exist in this vascular tissue.

The action of gamma-aminobutyric acid (GABA) and the GABAA-receptor agonists muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP) were studied at the single-channel level in outside-out membrane patches from cultured chick cerebral neurons. All agonists activated channels with multiple-conductance states. The main-state conductance activated by all agonists had a value around 26 pS in symmetrical TRIS/Cl solutions. Subconductance states of around 13 pS and 18 pS were seen with application of each agonist. Muscimol and isoguvacine tended preferentially to activate subconductance states. Gating by all agonists was complex. Open-time distributions for main-state activity gated by GABA, isoguvacine and THIP were best described by the sum of two exponential curves with similar time constants. Muscimol-gated activity was best described by the sum of three exponentials indicating the presence of an additional longer open state. These results indicate that certain GABAA-receptor agonists are capable of preferentially activating subconductance states.

1. Single Nae-activated K+ channels (KNa) were investigated by means of the inside-out patch clamp technique in ventricular myocytes isolated from the guinea-pig heart.2. Nae-activated K+ channels were observed at very low density (<9 % of patches Distributions of burst duration, between burst duration and openings within bursts were best described by the sum of two exponentials. Lowering [Na+]i decreased the burst duration and the duration of openings within burst. 6. These observations show that the Na+-activated K+ channel from guinea-pig ventricular myocytes has complex gating and bursting behaviour.Of the wide variety of potassium channels that are found in cell membranes, several show a high unit conductance of between 100 and 200 pS. A channel of this type activated by Nae (KNa) at the intracellular surface has been identified in both neuronal and myocardial cells at the whole-cell and single-channel level

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