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Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium ([K.sub.v]) channels, but are distinct in that they are gated solely by calcium ([Ca.sup.2+]), not voltage. For [K.sub.v] channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK9 channels. In this study, the divalent ions cadmium ([Cd.sup.2+]) and barium ([Ba.sup.2+]), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity, filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that [Cd.sup.2+] applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. [Cd.sup.2+] and MTSEA modify with similar rates whether the channels are open or closed. In contrast, [Ba.sup.2+] applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, [Ba.sup.2+] is trapped in SK2 channels when applied to open channels that are subsequently closed. [Ba.sup.2+] pre-block slows MTSEA modification of A384C in open but not in closed ([Ba.sup.2+]-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels.

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gatecl potasim (Kv) channels, but are distinct in that they are gated solely by calcium (Ca2+), not voltage. For Kv channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd2+) and barium (Ba2+), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd2+ applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd2+ and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba2+ applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba2+ is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba2+ pre-block slows MTSEA modification of A384C in open but not in closed (Ba2+- trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself. [ABSTRACT FROM AUTHOR]

Reports on the mediation of calmodulin binding by electrostatic interaction between residues. Rescue of surface expression by a mutant lacking the ability to bind calcium; Restoration of channel activity by application of purified recombinant; Requirement for gating.

Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells.