Chris T. Bond

Small-conductance, calcium-activated potassium channels from mammalian brain

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance,calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Mechanism of calcium gating in small-conductance calcium-activated potassium channels

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaption) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel α-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the α-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

Calcium-activated potassium channels expressed from cloned complementary DNAs

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

Altered Expression of Small-Conductance Ca2+-Activated K+ (SK3) Channels Modulates Arterial Tone and Blood Pressure

&NA; The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin‐sensitive, small‐conductance, Ca2+‐activated K+ channels may play an important role in active endothelium‐dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild‐type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohisto chemistry. In whole‐cell patch‐clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure‐ and phenylephrine‐induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure. (Circ Res. 2003;93:124‐131.)

Small Conductance Ca2+-Activated K+ Channel Knock-Out Mice Reveal the Identity of Calcium-Dependent Afterhyperpolarization Currents

Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+ (SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP.

EPISODIC ATAXIA RESULTS FROM VOLTAGE-DEPENDENT POTASSIUM CHANNELS WITH ALTERED FUNCTIONS

Episodic ataxia (EA) is an autosomal dominant human disorder that produces persistent myokymia and attacks of generalized ataxia. Recently, familial EA has been linked to the voltage-dependent delayed rectifier, Kv1.1, on chromosome 12. Six EA families have been identified that carry distinct Kv1.1 missense mutations; all individuals are heterozygous. Expression in Xenopus oocytes demonstrates that two of the EA subunits form homomeric channels with altered gating properties. V408A channels have voltage dependence similar to that of wild-type channels, but with faster kinetics and increased C-type inactivation, while the voltage dependence of F184C channels is shifted 20 mV positive. The other four EA subunits do not produce functional homomeric channels but reduce the potassium current when coassembled with wild-type subunits. The results suggest a cellular mechanism underlying EA in which the affected nerve cells cannot efficiently repolarize following an action potential because of altered delayed rectifier function.

Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and causes hypertension.

Background— It has been proposed that activation of endothelial SK3 (KCa2.3) and IK1 (KCa3.1) K+ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1−/−/SK3T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1−/−/SK3T/T (+doxycycline) mice abolished endothelial KCa currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1−/−/SK3T/T mice partially restored EDHF- and nitric oxide–mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1+/+/SK3T/T (+doxycycline) mice to normotensive levels. Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial KCa channels may represent novel therapeutic targets for the treatment of hypertension.

Small-conductance calcium-activated potassium channels.

ABSTRACT: SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetetive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long‐lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike‐frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin‐sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin‐insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin‐sensitive and apamin‐insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co‐injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co‐expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4‐5 or 4‐5‐4‐5, but not when they were connected 4‐4‐5‐5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4‐5‐4‐5. Further, the relative order of subunits within the channel contributes to their functional properties.

SK channels in excitability, pacemaking and synaptic integration

Small conductance calcium-activated potassium channels link elevations of intracellular calcium ions to membrane potential, exerting a hyperpolarizing influence when activated. The consequences of SK channel activity have been revealed by the specific blocker apamin, a peptide toxin from honeybee venom. Recent studies have revealed unexpected roles for SK channels in fine-tuning intrinsic cell firing properties and in responsiveness to synaptic input. They have also identified specific roles for different SK channel subtypes. A host of Ca2+ sources, including distinct subtypes of voltage-dependent calcium channels, intracellular Ca2+ stores and Ca2+-permeable ionotropic neurotransmitter receptors, activate SK channels. The macromolecular complex in which the Ca2+ source, SK channels and various modulators are assembled determines the kinetics and consequences of SK channel activation.