| Since their molecular identification, small conductance calcium-activated potassium channel have emerged as important regulators of membrane excitability in central nervous system and in neuronal tissues. Three isoforms of the SK channel exist: SK1--SK3. These channels are subjected to multi-faceted regulation from the transcriptional to post-translational levels. The members of the SK channel family along with IK channel share a great deal of sequence conservation, and all of them use calmodulin as a beta subunit that confers Ca2+ sensing activity. One consequence of multi-level regulation of SK channels is control of cellular localization.; We discovered a novel genetic tool for silencing SK and IK channels, SK3-1C. This naturally occurring transcript has been extensively characterized, and the domain responsible for the ability of SK3-1C to inhibit native currents in the adrenal chromaffin cell carcinoma (pheochromacytoma) cell line, PC12, has been identified. The extensive conservation in the dominant inhibitory sequence, or DIS, indicates that SK3-1C can silence other members of the SK channel family. Whole cell patch clamp experiments revealed that SK3-1C is able to silence the entire SK channel family as well as the IK channel. Immunocytochemistry using and anti-SK3 antibody and confocal microscopy has determined SK3 channel localization in PC12 cells transfected with SK3-1C is dramatically altered.; The presence of SK3 subunits in PC12 cells indicated that SK channels may play an important role in normal adrenal tissue. Immunohistological and immunocytological experiments indicate that SK3 is the predominant channel found in the adrenal medulla, while SK2 appears to predominate in the adrenal cortex. SK channels also appear to be expressed in adrenomedullary chromaffin cells (ACCs) that also express the enzyme phenylethanolamine-N-methyl transferase (PNMT), which is a marker for epinephrine secreting cells. SK channels tend to reduce the excitability of ACCs, as reduction in activity of SK channels using inhibitor compounds increases the action potential firing rate and the opener NS309 slows down firing. ACCs from mice overexpressing SK2 and SK3 exhibit reduced excitability comparable to NS309 treatment, though the effects of overexpressing SK3 are much more severe. Ca2+ influx induced by slight increases in external K+ concentrations were also enhanced by SK blockade and reduced by NS309. Lastly, preliminary data from analysis of catecholamines in cell culture medium from ACCs treated with high K+ and SK channel pharmacological agents is encouraging as a potential method to analyze catecholamine secretion without the difficulties in controlling experiments done in live animals. Throughout the study of SK channels and the adrenals, several future directions for study of SKs in this tissue have emerged. |
