(J-393) Chemical Compound Screening for BK Ion Channel

Ion channels are regulators of fundamental cellular processes such as cellular communication, making them essential for life. They are often pharmaceutical targets in medicine because of their crucial role within muscular systems, the nervous system, and neuron excitability. The big conductance potassium ion channel (BK channel) regulates membrane excitability and intracellular calcium homeostasis through a negative feedback loop. This transmembrane channel is ubiquitously expressed within the human body, with distinct functions for each tissue type. The BK channel regulatory mechanisms begin following activation. When the cellular membrane depolarizes, the intracellular calcium concentration increases causing hyperpolarization and the release of potassium (K+) ions from internal stores. Which closes calcium voltage-dependent channels.  This mechanism controls the K+ transport in and out of the cell. In humans, KCNMA1 (Slo1) encodes the BK channel. There are three different domains within the Slo1 subunit that undergo splicing to create various BK isoforms. The three structural domains are the pore-gating domain, voltage-sensing domain, and cystolic domain. Mutations and dysfunctions within the genes coding the BK channel can cause a wide range of diseases and conditions throughout the nervous system and cardiovascular systems. Such as epilepsy, mental retardation, hypertension, hypoxia, and stroke. Finding chemical compounds that affect the BK channel current or activation can aid in the treatment of various health conditions and diseases.

Using mature Xenopus laevis (African clawed frog) oocytes were injected with mRNA of Wild-Type (WT) mSlo1. This was completed to allow the oocytes to express the BK ion channel, so the channel can be recorded and screened to identify channel modulators. Approximately 2-3 days following the injection, the oocyte membrane was stripped and placed in a bath solution underneath a microscope. A glass micro-pipette was prepared with a wire in an electrolytic solution and positioned horizontally in close proximity to the oocyte. Using the patch clamp technique, a tight giga-ohm seal was then created within the cell membrane. The pipette was then pulled away from the cell containing a patch of the membrane, so the isolated BK ion channel can be recorded. Following the formation of a patch, the resistance of the membrane was expressed in Giga-ohms, and the current was visible on the current graph, being at least 3 A. Indicating a successful patch and RNA injection. Using a perfusion system, the patch was initially perfused with a zero calcium/ zero magnesium solution as the baseline control. The current was recorded from -30mV to 250 mV, and run three different times forming a new patch for each trial. Following three screenings with the control solution, chemical compounds were screened using the same process. The first drug screened was MolPort-0090651-309 (1- < 2-[3-(naphthalen-2-vl)-6-oxo-1,6-dihydropyridazin-1-yllacetyl\piperidine-4-carboxylic acid), also denoted as B2. Following this drug screen, B7, MolPort-001-513-013 (N-(4-< [2-(4-hydroxyphenyl)-1,3-dioxo-2,3-dihydro-1H-isoindol-5-vlloxy?phenyl)cyclohexanecarboxamide) was also tested using the same procedure.

In order to identify chemical compounds that positively fit with the BK channel, a docking target computer site tested the compound structure with specific intracellular sites on the channel indicating a fit, meaning they could potentially affect channel function.  The control WT current caused the channel to open at approximately 100 mV. Following the complete drug screen for B2 the conductance-voltage (G-V) curve was analyzed to determine if there was a shift from the control. Results indicated that the channel open probability was not affected in the presence of B2 in comparison to the control. Although there was no G-V shift, the B2 drug did cause a slight increase in the current (Figure 1B). But not enough to determine definitively that there is a current increase due to B2, because GV is not affected (Figure 1C). After the B7 complete drug screen, there was a minimal difference between the B7 drug and the control for the current and G-V.

If there was a shift in G-V from the control solution, this would mean the activation of BK channel is harder to close (shifting to more negative voltage values) or easier to open by shifting to more positive voltages. Finding a chemical compound that affects the intrinsic opening of the BK channel means various isoforms of the BK channel that cause abnormalities seen in vascular tone and neurological diseases can allow for the development of ion therapies to correct these conditions. As an example, in the cardiovascular system, the hyperpolarization and closing of calcium-dependent channels cause vasodilation, which is critical for endothelial function. This means the BK channel has a direct role in the regulation of blood pressure. Blood pressure is measured to determine the parameters of vital body functioning. Limiting hypertension decreases damage to one's arteries and heart, preventing the occurrence of heart attacks and strokes. Although the B2 and B7 drugs were unsuccessful in affecting the BK channel, upon more research and chemical compound screening the discovery of channel modulators will be available to provide various options for treatment for several cardiovascular conditions.