Intermediate‐conductance calcium‐activated potassium channels in enteric neurones of the mouse: pharmacological, molecular and immunochemical evidence for their role in mediating the slow afterhyperpolarization

Abstract

Calcium-activated potassium channels are critically important in modulating neuronal cell excitability. One member of the family, the intermediate-conductance potassium (IK) channel, is not thought to play a role in neurones because of its predominant expression in non-excitable cells such as erythrocytes and lymphocytes, in smooth muscle tissues, and its lack of apparent expression in brain. In the present study, we demonstrate that IK channels are localized on specific neurones in the mouse enteric nervous system where they mediate the slow afterhyperpolarization following an action potential. IK channels were localized by immunohistochemistry on intrinsic primary afferent neurones, identified by their characteristic Dogiel type II morphology. The slow afterhyperpolarization recorded from these cells was abolished by the IK channel blocker clotrimazole. RT-PCR and western analysis of extracts from the colon revealed an IK channel transcript and protein identical to the IK channel expressed in other cell types. These results indicate that IK channels are expressed in neurones where they play an important role in modulating firing properties.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells

“…Although KCa3.1 channel activation has been documented in cells of the enteric and myenteric nervous systems (36,37), several lines of evidence now support the expression of KCa3.1 channels in Purkinje cells, including single cell RT-PCR, KCa3.1 immunolabel, single channels with intermediate conductance, and macropatch recordings of KCa currents with a pharmacological profile that is unique to KCa3.1 channels (3, 7). Moreover, Cav3.2 Ca 2+ channels colocalize and associate with KCa3.1 channels to provide Ca 2+ -dependent regulation at the nanodomain level to control temporal summation of parallel fiber EPSPs.…”

Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40

“…A characterized commercial anti-rat Cx43 (Zymed) antibody raised in rabbit was also used. Specificity of the IK1 (IK38/6), Cx37, Cx40, and Cx43 antibodies has been determined previously by Western blot and immunolabeling of transfected cells (37,42,44).…”

“…Secondary antibodies were incubated for 1 h at 22°C (Cy3 conjugated anti-rabbit or anti-goat immunoglobulins, 1:100; Jackson Immunoresearch Laboratories) in 0.01% Triton X-100 in PBS, and vessels were mounted in buffered glycerol. IK1 (SK4) antibodies were raised in rabbits against amino acids 2-17 of rat (37) and antibodies against rat Cx37 and Cx40 raised in sheep (42,44). A characterized commercial anti-rat Cx43 (Zymed) antibody raised in rabbit was also used.…”

“…Charybdotoxin was dissolved in PBS containing 0.1% BSA. Cx-mimetic peptides 40 Gap27 and 37,43 Gap27 (SRPTEKNVFIV, SRPTEKTIFII, respectively) were synthesized by the Australian Cancer Research Foundation Biomolecular Resource Facility, John Curtin School of Medical Research; purity Ͼ97%. All other drugs were made up as ϫ1,000 stock solutions in distilled water and diluted into Krebs solution.…”

“…Combined charybdotoxin and apamin abolished vasomotion and depolarized and constricted vessels, even in absence of endothelium. Separately, 37,43 Gap27 and 40 Gap27 abolished vasomotion. Extensive myoendothelial gap junctions (3 per endothelial cell) composed of connexins 37 and 40 connected the endothelial cell and SMC layers.…”