The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

Area of Science:

• Cellular physiology
• Ion channel function
• Biophysics

Background:

• Previous research identified a 16-pS K+ channel (KCca) in the basolateral membrane responsible for acetylcholine-induced K+ conductance.
• The precise regulatory mechanisms of this channel remained to be fully elucidated.

Purpose of the Study:

• To investigate the biophysical properties and regulation of the 16-pS K+ channel (KCca) in excised membrane patches.
• To determine the role of calcium and ATP in controlling channel activity.

Main Methods:

• Excised inside-out patch-clamp electrophysiology on basolateral membrane patches.
• Voltage-ramp analysis to assess channel rectification.
• Dose-response studies with calcium to determine sensitivity.
• Application of ATP and non-metabolizable analogs (AMP-PNP) to assess regulatory effects.
• Treatment with alkaline and acidic phosphatases to investigate phosphorylation status.

Main Results:

• The 16-pS K+ channel exhibited inward rectification over a broad voltage range.
• Channel activity was highly sensitive to cytosolic calcium, with half-maximal activation at 330 nmol/l and a Hill coefficient of three.
• A slow run-down of channel activity was observed over time, which could be reversed by adding ATP.
• Non-metabolizable ATP analog AMP-PNP inhibited channel activity, while ATP itself refreshed it.
• Phosphatase treatment significantly reduced channel activity, suggesting a role for phosphorylation.

Conclusions:

• The 16-pS K+ channel is directly regulated by cytosolic calcium levels.
• ATP-dependent regulation, likely involving phosphorylation, plays a crucial role in maintaining channel function.
• High concentrations of non-metabolizable ATP analogs can inhibit this potassium channel, similar to other K+ channels.