We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

A cDNA (CSMK1) encoding a delayed rectifier K+ channel of the K,1.2 dass was cloned from canine colonic circular smooth muscle and expressed in Xenopus oocytes. These channels appear to be uniquely expressed in gastrointestinal muscles and may participate in the electrical slow wave activity. Functional expression of CSMK1 in Xenopus oocytes demonstrated a K+ current that activated in a voltage-dependent manner upon depolarization. This current was highly sensitive to 4-aminopyridine (IC5o, 74 pM). A

Delayed rectifier K+ channels are involved in the electrical activity of all excitable cells. The relationship between native K+ currents recorded from these cells and cloned K+ channel cDNAs has been difficult to ascertain partly because of contradictions in pharmacological characteristics between native and expressed currents. Through the study of the charybdotoxin (CTX) pharmacology of two cloned smooth muscle delayed rectifier K+ channels (cKv 1.2 and cKv1.5) expressed in oocytes, evidence for heterotetramer formation was obtained. We have shown that the presence of even a single CTX-insensitive subunit renders the heterotetrameric channel insensitive to CTX. The two K+ channel clones differ in an amino acid at the mouth of the pore region, which may be in a position to block the access of CTX to its binding site and hence determine CTX sensitivity of the heterotetrameric channel. These results may explain discrepancies reported between native and cloned smooth muscle K+ channels.

1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier K,12 K+ channel expressed in oocytes was investigated at room temperature (22°C) and physiological temperature (34°C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 uM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a I s step.3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34°C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34°C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced.

1 Single smooth muscle cells were isolated from the rabbit portal vein and the human mesenteric artery and whole cell currents recorded at room temperature from either cell type by the whole cell voltage clamp technique. 2 In the rabbit portal vein cells addition of 10pUM BRL 38227 induced a quasi-instantaneous, voltageinsensitive and time-independent current which had a reversal potential of -75 mV under experimental conditions where the calculated EK was -83 mV. 3 Cells were held at 0 mV and BRL 38227 was added cumulatively to construct a dose-response relationship. BRL 38227 (0.03-10giM) caused a dose-dependent outward shift in the holding current with an EC50 of 1.3gUM. 4 BRL 38227 (10M) had no effect on the delayed rectifier K+ current measured in the presence of 5mM tetraethylammonium and no effect on the Ca2 -activated K+ current measured in the presence of 5 mM 4-aminopyridine. Similarly BRL 38227 had no effect on the Ca21 current. 5 The BRL 38227-induced current was blocked by glibenclamide (10gM) and phentolamine (100UM), specific blockers of the ATP-sensitive K+ current in single cells. 6 In human isolated mesenteric artery cells, BRL 38227 (10pM) induced a glibenclamide-sensitive current similar to, but smaller than, that observed in the rabbit portal vein. 7 We conclude that in these cells, BRL 38227 activates a potassium conductance which has the electrophysiological and pharmacological characteristics of ATP-sensitive K+ channels.